Method for producing polyhydroxyalkanoic acid using alginic acid as a carbon source

Abstract

To provide a method for producing polyhydroxyalkanoic acid (PHA) using alginic acid as a carbon source, and to provide a microorganism capable of synthesizing PHA using alginic acid as a carbon source.SOLUTION: According to the present invention, provided is a method for producing PHA, comprising culturing a microorganism having alginate assimilation ability and PHA synthesis ability and extracting PHA from the culture, in a medium containing either selected from the group consisting of alginic acid and alginic acid decomposition products. According to the invention, provided is a bacterial strain of the genus Cobetia having 16S rRNA gene comprising: (a) a specific base sequence; or (b) a base sequence having 90% or more base sequence identity to the specific base sequence.SELECTED DRAWING: Figure 5A

Description

[Technical field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid using alginic acid as a carbon source. [Background technology]

[0002] Biodegradable plastics are polymeric materials that have the same properties and functions as ordinary plastics when not in contact with microorganisms, but when placed in the natural environment or in a composting device, they are assimilated by microorganisms and eventually decomposed into carbon dioxide and water. There are two types of biodegradable plastics: biodegradable petroleum-derived plastics that use fossil resources as raw materials, and biodegradable bioplastics that use biomass as raw materials. As the increase in carbon dioxide emissions and global warming become problems, there is a demand for materials that use biomass as raw materials, such as bioplastics, that ultimately decompose into water and carbon dioxide, and do not accumulate in the environment.

[0003] The Japan Bioplastics Association states that a product must have a bioplastic content of 25% or more by mass in order to meet the criteria for identifying and labeling biomass plastics. Products with a higher bioplastic content have a lower environmental impact. Examples of bioplastics made from 100% biomass raw materials include polylactic acid and polyhydroxyalkanoic acid (PHA).

[0004] PHA is an aliphatic polyester synthesized by microorganisms, and has a physiological role as a carbon source and energy compound. It accumulates in cells when a carbon source is present under nutrient-depleted conditions in which nitrogen and phosphorus sources are insufficient. Polyhydroxybutanoic acid (P(3HB)), one of the most researched PHAs, was discovered in Bacillus megaterium by Dr. Lemoigne in 1925, and its synthesis by more than 300 types of bacteria, including hydrogen bacteria and blue-green algae, has since been reported. This P(3HB) has a brittle nature (breaking elongation 5%) and was considered to be a commercially inferior material compared to polypropylene. However, since Wallen and Rohwedder discovered a hydroxyalkanoic acid (HA) unit different from the 3-hydroxybutanoic acid unit (3HB) in 1974, various HAs have been reported, and currently more than 150 types of monomer units have been reported. In addition, various studies have shown that the physical properties of PHA can be improved by incorporating monomer units other than 3HB to form a copolymer (Non-Patent Documents 1-6).

[0005] Microbial biosynthesis of PHA using various organic substances as carbon sources has been reported. For example, Patent Document 1 discloses a method for producing polyhydroxyalkanoic acid by a microorganism, comprising culturing a microorganism belonging to the genus Cupriavidus, Ralstonia, or Alcaligenes, which has the ability to assimilate lignin derivatives and / or their polyhydroxyalkanoic acid biosynthesis intermediates, in a medium containing at least one substance selected from the group consisting of lignin derivatives and their polyhydroxyalkanoic acid biosynthesis intermediates as a carbon source, and recovering polyhydroxyalkanoic acid from the microbial cells. Patent Document 2 discloses a method for producing a polyhydroxyalkanoic acid copolymer, which comprises: (i) culturing a transformant in which a polyhydroxyalkanoic acid polymerase mutant gene and a leucine metabolism gene derived from a Clostridium microorganism have been introduced into a microorganism in the presence of a sugar; and (ii) collecting a polyhydroxyalkanoic acid copolymer containing 3-hydroxybutanoic acid and 3-hydroxy-4-methylvaleric acid as monomer units from the resulting culture, wherein the sugar is a monosaccharide (glucose).

[0006] Patent Document 3 discloses a method for producing polyhydroxyalkanoic acid by using a microorganism, which is obtained by polymerizing monomer units containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and is characterized in that the method uses fats and oils containing 41% by weight or more of lauric acid as a constituent fatty acid and / or fatty acids as a carbon source, and cultures the microorganisms while adjusting the oxygen transfer rate so that the average hourly productivity of polyhydroxyalkanoic acid from the start of culture is 1.5 g / L / h or more. Other examples include a method for producing polyhydroxyalkanoic acid or a polyhydroxyalkanoic acid copolymer by a microorganism using sugars (glucose, fructose, sucrose), fats and oils, or fatty acids as a carbon source (Patent Documents 4 to 7); a method for producing polyhydroxyalkanoic acid by fermentation using a microorganism, characterized in that the carbon source contains a carboxylic acid having four carbon atoms and / or an alcohol having four carbon atoms and a derivative thereof (Patent Document 8); a method for producing a microbial polyester, characterized in that a microorganism capable of producing a polyester is cultured in a medium containing 1-hexene as the sole carbon source (Patent Document 9); and a method for producing polyhydroxyalkanoic acid, characterized in that a microorganism capable of growing by assimilating acetic acid or a salt thereof and of producing and accumulating polyhydroxyalkanoic acid is cultured in a medium containing acetic acid or a salt thereof as the sole carbon source (Patent Document 10).

[0007] PHA production using sugars and alcohols extracted from edible raw materials such as corn and sugarcane competes with food and causes prices to rise. Therefore, research into utilizing unused biomass is expected to be a technology that places a low burden on the global environment. In recent years, seaweed has attracted worldwide attention as one of the unused biomass. In Japan, a large amount of seaweed is discarded at the seaweed production and processing stage, and about 60% of the harvested amount is discarded in wakame processing (Non-Patent Document 7). This seaweed waste must be treated as industrial waste, which is a heavy burden for fishermen and companies, and illegal dumping of seaweed waste is also a problem. Seaweed belonging to brown algae such as kombu and wakame has been reported to contain 20-40% alginic acid per dry weight (Non-Patent Document 8). In addition, the content of alginic acid in seaweed is almost constant throughout the year (Non-Patent Document 9). For this reason, alginic acid is a promising raw material as a marine resource. However, although there have been reports of PHA biosynthesis using cellulose and mannitol, which are major components of seaweed other than alginic acid, as carbon sources (Non-Patent Documents 10 to 12), there have been no reports of PHA biosynthesis using alginic acid as a carbon source. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2015-077103 A [Patent Document 2] JP 2015-33 A [Patent Document 3] JP 2013-9627 A [Patent Document 4] WO2012 / 102371 [Patent Document 5] WO2009 / 147918 [Patent Document 6] JP 2009-225662 A [Patent Document 7] Patent Application No. 2006-238032 [Patent Document 8] JP 2009-225775 A [Patent Document 9] JP 2001-178487 A [Patent Document 10] JP 2001-178484 A [Non-patent literature]

[0009] [Non-Patent Document 1] Kenichiro Matsumoto, Yoshiharu Doi, Biosynthesis of Biodegradable Polyesters: Current Status and Prospects, Journal of the Society of Organic Synthetic Chemistry, Vol. 61, No. 5, pp. 489-495, 2003 [Non-Patent Document 2] K. Sudesh, T Iwata, Sustainability of Biobased and Biodegradable Plastics, Clean 2008, 36(5-6), 433-442 [Non-Patent Document 3] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Prog. Polym. Sci., 25(10), 1503-1555, (2000) [Non-Patent Document 4] Tadahisa Iwata, Structure, properties and biodegradability of microbial polyesters, Journal of the Crystallographic Society of Japan, 55, 188-196(2013) [Non-Patent Document 5] Wallen LL, Rohwedder WK, Poly-β-hydroxyalkanoate from activated sludge, Environ Sci Technol 8: 576-579 (1974) [Non-Patent Document 6] JML Dias, PC Lemos, LS Serafim, C. Oliveria, M. Eiroa, MGE Albuquerque, AM Ramos, R. Oliveira, M. A M. Reis, Recent Advances in Polyhydroxyalkanoate Production by Mixed Aerobic Culture: From the Substrate to the Final Product, Macromol. Biosci., 6(11), 885-906 (2006) [Non-Patent Document 7] Shinichiro Fujii, Takashi Koenaga, Material flow analysis and zero emission technology in the wakame seaweed processing industry, Journal of Environmental Science 13(5), 586-592, (2000) [Non-Patent Document 8] Hiroyuki Takeda, Fuminori Yoneyama, Shigeyuki Kawai, Wataru Hashimoto and Kousaku Murata, Bioethanol production from marine biomass alginate by metabolically engineered bacteria, Energy Environ. Sci., 4, 2575-2581, (2011) [Non-Patent Document 9] T. Kimura, K. Ueda, R. Kuroda, T. Akao, N. Shinohara, T. Ushirokawa, A. Fukagawa and T. Akimoto, Nippon Suisan Gakkaishi, The seasonal variation in polysaccharide content of brown alga akamoku Sargassum horneri collected off Oshima Island (Fukuoka Prefecture), Nippon Suisan Gakkaishi, 73(4), 739-744, (2007) [Non-Patent Document 10] LE Alva Munoz, MR Riley, Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans, Biotechnol. Bioeng., 100(5), 882-888, (2008) [Non-Patent Document 11] F. Cerrone, R. Davis, ST Kenny, T. Woods, A. O'Donovan, VK Gupta, M. Tuohy, RP Babu, P. O'Kiely, K. O'Connor, Use of a mannitol rich ensiled grass press juice (EGPJ) as a sole carbon source for polyhydroxyalkanoates (PHAs) production through high cell density cultivation, Bioresour. Technol., 191, 45-52, (2015) [Non-Patent Document 12] M. Yamada, A. Yukita, Y. Hanazumi, Y. Yamahata, H. Moriya, M. Miyazaki, T. Yamashita, H. Shimoi, Poly(3-hydroxybutyrate) Production using mannitol as a sole carbon source by Burkholderia sp. AIU M5M2 isolated from a marine environment, Fisheries Science, 84(2), 405-412, (2018) Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, PHA biosynthesis using sugars, alcohols, and other carbon sources has been reported in the past. However, because these carbon sources compete with food, there has been a demand for the development of a production method for bioplastics using unused biomass as a raw material.

[0011] Alginic acid is abundant in seaweed and is a promising raw material as a marine resource. However, there have been no reports on the biosynthesis of PHA using alginic acid as a carbon source. Therefore, the present invention aims to provide a method for producing PHA using alginic acid as a carbon source, and to provide a microorganism capable of synthesizing PHA using alginic acid as a carbon source. [Means for solving the problem]

[0012] As a result of extensive research to solve the above problems, the inventors discovered a microorganism capable of synthesizing PHA using alginic acid as a carbon source, and a method for producing PHA using this microorganism, thereby completing the present invention. Specifically, the present invention provides the following [1] to

[13] . [1] A method for producing polyhydroxyalkanoic acid (PHA), comprising culturing a microorganism capable of utilizing alginic acid and synthesizing PHA in a culture medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture. [2] The manufacturing method described in [1], which includes a preliminary step of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or acid. [3] The method according to [1] or [2], wherein the microorganism is selected from marine bacteria belonging to the Gammaproteobacteria class. [4] The method according to any one of [1] to [3], wherein the microorganism is any one selected from bacteria belonging to the genus Cobetia. [5] The method according to any one of [1] to [4], wherein the microorganism is any one selected from the following 1) or 2). 1) Microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759. 2) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b): (a) A base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2. [6] The method according to any one of [1] to [5], wherein the microorganism is an isolated microorganism, a deposited microorganism, or a genetically modified microorganism. [7] The method according to any one of [1] to [6], wherein the concentration of alginic acid or alginic acid decomposition product in the medium is in the range of 0.5% by mass to 20% by mass. [8] The method according to any one of [1] to [7], wherein the concentration of sodium chloride in the medium is in the range of 0.5% by mass to 20% by mass. [9] The method according to any one of [1] to [8], wherein the PHA contains a 3-hydroxybutanoic acid monomer unit.

[10] The method according to [9], wherein the PHA is poly(3-hydroxybutanoic acid).

[11] A step of obtaining polyhydroxyalkanoic acid (PHA) by carrying out the method according to any one of [1] to

[10] ; A method for producing a bioplastic product, comprising the step of obtaining a plastic product containing 25% by mass or more of the PHA obtained in the above step.

[12] Microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759.

[13] A bacterium of the genus Covetia having a 16S rRNA gene containing the following nucleotide sequence (a) or (b): (a) A base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2. Effect of the Invention

[0013] According to the present invention, a method for producing a bioplastic using alginic acid as a carbon source can be provided. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the biosynthetic pathway of PHA. [Diagram 2] Fig. 2A shows an example of the state of culture of a natural sample in seawater. Fig. 2B shows an example of the state of culture of a natural sample in agar medium (ZoBell 2216E seawater medium). [Figure 3-1] Figure 3-1 shows some of the results of culture performed on 121 samples from the 171 strains obtained in the primary screening. [Figure 3-2] Figure 3-2 shows some of the results of culture performed on 121 samples from the 171 strains obtained in the primary screening. [Figure 4A] FIG. 4A shows the results obtained by GC-MS for the 5-28-6-1 strain. [Figure 4B] FIG. 4B shows the results obtained by GC-MS for the 5-11-6-3 strain. [Figure 5A] FIG. 5A shows the results of 1H-NMR analysis of the 5-28-6-1 strain. [Figure 5B] FIG. 5B shows the results of 1H-NMR analysis of the 5-11-6-3 strain. [Figure 6] Figure 6 shows the nucleotide sequences of the 16S rRNA gene. A: strain 5-28-6-1, B: strain 5-11-6-3. [Figure 7-1] Figure 7-1 shows an alignment of the nucleotide sequences of the 16S rRNA gene. [Figure 7-2] Figure 7-1 shows the continuation of the alignment of 16S rRNA gene base sequences. [Figure 8] FIG. 8 shows the Gram staining images of the microorganisms strains 5-28-6-1 and 5-11-6-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits.

[0016] The present invention provides a method for producing polyhydroxyalkanoic acid (PHA), which comprises culturing a microorganism capable of utilizing alginic acid and synthesizing PHA in a medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture.

[0017] The production method of the present invention can be carried out using a microorganism having the ability to utilize alginic acid and synthesize PHA. The ability to utilize alginic acid is the property of being able to grow using alginic acid as a carbon source. The ability to synthesize PHA is the property of being able to biosynthesize PHA. It can be said that a microorganism having the ability to utilize alginic acid and synthesize PHA is a microorganism having the ability to synthesize PHA using alginic acid as a carbon source. The microorganism having the ability to utilize alginic acid and synthesize PHA may be a microorganism that can utilize alginic acid decomposition products in addition to alginic acid and synthesize PHA.

[0018] Whether or not a microorganism has the ability to utilize alginic acid can be determined by culturing the microorganism in a medium containing alginic acid as the sole carbon source and determining whether or not the microorganism grows. For example, a test microorganism is cultured in a medium containing sodium alginate at a concentration of 1% by mass as the only carbon source under conditions suitable for the growth of the test microorganism (temperature, pH, oxygen, sodium salt concentration, etc.), and if the test microorganism grows, it can be determined that the test microorganism has the ability to utilize alginic acid. The growth of the test microorganism can be measured by the turbidity method, the plate culture method, the dry cell weight method, etc.

[0019] Whether a microorganism has the ability to synthesize PHA can be determined by culturing it in a nitrogen- or phosphorus-limited medium and determining whether or not it synthesizes PHA. For example, if a test microorganism is cultured in a nitrogen-source-limited inorganic salt medium containing 1-2% glucose as the sole carbon source under conditions suitable for the growth of the test microorganism and the test microorganism accumulates PHA in the body, the test microorganism can be determined to have the ability to synthesize PHA. The accumulation of PHA can be confirmed by Nile Red or Nile Blue A staining method, and can also be determined by observing PHA granules in cells using a transmission microscope, NMR analysis of a chloroform extract obtained from dried cells, gas chromatography or gas chromatography mass spectrometry of a chloroform extract treated with ethanolysis, etc. In addition, when the accumulated PHA is PHB, the accumulation of PHB can be determined by converting it to crotonic acid using sulfuric acid and then analyzing it using high performance liquid chromatography.

[0020] The microorganism having the ability to utilize alginic acid and synthesize PHA may be a halophilic marine bacterium belonging to the class Gammaproteobacteria. The class Gammaproteobacteria belongs to the phylum Proteobacteria of eubacteria. Bacteria belonging to the class Gammaproteobacteria are gram-negative bacteria. Marine bacteria are bacteria that live in the ocean and can grow well in seawater. Halophilic bacteria are bacteria that can grow well in a sodium salt concentration of about 0.5 to 20%.

[0021] The microorganism having the ability to utilize alginic acid and synthesize PHA may be a bacterium belonging to the genus Cobetia, such as Cobetia marina, Cobetia pacifica, Cobetia amphilecti, Cobetia litoralis, and an undescribed species that is inferred to belong to the genus Cobetia from the 16S rDNA sequence. The microorganisms having the ability to utilize alginic acid and synthesize PHA of the present invention can be selected from the genus Cobetia, preferably Cobetia marina and its closely related species (including undescribed species), more preferably from the species to which the 5-28-6-1 and 5-11-6-3 strains described in the Examples below belong, and it is particularly preferable to use the 5-28-6-1 and 5-11-6-3 strains. Cobetia marina was previously classified as Arthrobacter marinus, Pseudomonas marina, Dalya marina, and Halomonas marina (Arahal DR, et al., (December 2002) Systematic and Applied Microbiology. 25 (2): 207-11).

[0022] In one embodiment of the present invention, the above-mentioned microorganism having the ability to utilize alginic acid and synthesize PHA can be any one selected from the following 1) or 2). 1) Microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759. 2) A microorganism having a 16S rRNA gene containing the following base sequence (a) or (b): (a) A base sequence set forth in SEQ ID NO: 1 or 2. (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0023] The above-mentioned microorganism may be a microorganism having a 16S rRNA gene containing a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of base sequence identity to the base sequence set forth in SEQ ID NO: 1 or 2, and having the ability to utilize alginic acid and synthesize PHA. Furthermore, the above-mentioned microorganism may be a microorganism belonging to the genus Covetia having a 16S rRNA gene containing a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of base sequence identity to the base sequence set forth in SEQ ID NO: 1 or 2, and having the ability to utilize alginic acid and synthesize PHA. The microorganism may also be a microorganism belonging to the genus Covetia, which has a 16S rRNA gene containing a base sequence in which one or several bases have been substituted, inserted, deleted and / or added in the base sequence set forth in SEQ ID NO: 1 or 2, and has the ability to utilize alginic acid and synthesize PHA.

[0024] The base sequence can be analyzed by a conventional method. For example, a method for amplifying the 16S rRNA gene from the DNA of a microorganism includes, but is not limited to, a PCR method using a primer. The amplified product obtained by the PCR method can be subjected to a DNA sequencer or the like to analyze the base sequence.

[0025] Sequence identity is defined as the percentage of bases that are identical between two sequences after aligning the two sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Sequence identity can be determined by using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, ClustalX, or Megalign (DNASTAR) software.

[0026] As used herein, the range of "one or several" in "a base sequence in which one or several bases have been substituted, inserted, deleted and / or added" is not particularly limited, but means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0027] The microorganism may be an isolated microorganism, a deposited microorganism, or a genetically modified microorganism. An isolated microorganism is a microorganism isolated from a natural sample. Examples of natural samples include seawater, marine organisms, seaweed, brackish water, brackish organisms, rocks, soil, and sand. A natural sample can be cultured in any medium, such as ZoBell 2216E seawater medium, MS medium, Daigo IMK medium, etc., at 10°C to 42°C, for 24 to 72 hours, at pH 5 to 10, and a colony can be isolated. Before culturing using ZoBell 2216E seawater medium, MS medium, etc., the sample can be pre-cultured in filtered seawater. The isolated colonies can be cultured in an alginic acid-containing medium, and colonies that show proliferation in the alginic acid-containing medium and are judged to be synthesizing PHA can be selected. Nile Red is a hydrophobic dye that reacts with hydrophobic substances such as neutral lipids and PHA in the living body to turn red, and can be used to screen microorganisms capable of synthesizing PHA.

[0028] Furthermore, for PHA synthesis, PHA can be extracted from dried microbial cells with an organic solvent such as chloroform, and then precipitated by adding an organic solvent such as methanol or hexane, and screened using analytical techniques such as gas chromatography, liquid chromatography, NMR, IR, etc. Furthermore, in some cases, the strain of the isolated colony can be identified based on the 16S rDNA sequence or bacteriological properties.

[0029] The deposited microorganisms can be obtained by selecting microorganisms capable of alginate assimilation and PHA synthesis from the deposited microbial material using the same techniques as those described for the isolated microorganisms. For example, microorganisms capable of alginate assimilation and PHA synthesis can be selected from the deposited marine bacteria belonging to the class Gammaproteobacteria. Furthermore, microorganisms capable of alginate assimilation and PHA synthesis can be selected from the deposited microorganisms belonging to the genus Covetia. The genetically modified microorganism can be, for example, a microorganism in which an alginate decomposition enzyme gene, a PHA synthase gene, and / or genes related thereto have been modified.

[0030] The polyhydroxyalkanoic acid (PHA) for which the production method is provided by the present invention is a polyester known to accumulate in the bodies of certain microorganisms and has the following chemical formula: [ka] [wherein R may be the same or different and is a linear or branched alkyl group having 1 to 14 carbon atoms, and n is the number of repetitions and is an integer of 2 or more, preferably an integer of 100 or more, and preferably an integer of 100,000 or less]. Specific examples of PHAs include those having the following formula: [ka] In addition to polyhydroxybutanoic acid (P(3HB) or PHB) having the formula: Representative copolymers include poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] with 3-hydroxyvaleric acid units, poly(3-hydroxybutanoic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)] with 3-hydroxycaproic acid units, poly(3-hydroxybutanoic acid-co-3-hydroxypropionic acid) [P(3HB-co-3HP)] with 4-hydroxybutanoic acid units, poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)] with lactic acid units, and poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)] with glycolic acid units.

[0031] The biosynthetic pathway of PHA, P(3HB), has been well studied in Ralstonia eutropha, and it has been reported that two molecules of acetyl-CoA are condensed to acetoacetyl-CoA by β-ketothiolase (PhaA), which is reduced to (R)-3-hydroxybutyryl-CoA by NADPH-dependent acetoacetyl-CoA reductase (PhaB), and then polymerized by PHA synthase (PhaC) (Fig. 1) (Yamada, M. et al., Oleoscience, 5 (11), 523-532 (2005); K. Sudesh et al., Prog. Polym. Sci. 25 (2000) 1503-1555). In addition, the biosynthesis of 3HA with a medium chain length of 6-14 carbons has been studied in Pseudomonas bacteria and Aeromonas caviae, and it has been reported that the β-oxidation of fatty acids and the biosynthetic pathway are involved (Fig. 1) (Hiromi Matsuzaki et al., Journal of the Japan Oil Chemists' Society, 48(12), 1353-1363, 1999; T. Fukui et al., J. Bacteriol, 180(3), 667-673, 1998; T. Fukui et al., J. Bacteriol, 179(15), 4821-4830, 1997).

[0032] The polyhydroxyalkanoic acid (PHA) produced by the production method of the present invention can have a molecular weight of 200,000 to 2,000,000. The polyhydroxyalkanoic acid (PHA) for which the production method of the present invention provides can be one that contains 3-hydroxybutanoic acid monomer units. Examples of PHA that contain 3-hydroxybutanoic acid monomer units include poly(3-hydroxybutanoic acid-co-3-hydroxypropionic acid) [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)], poly(3-hydroxybutanoic acid-co-3-hydroxycaproic acid) [P(3HB-co-3HHx)], poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-lactic acid) [P(3HB-co-LA)], and poly(3-hydroxybutanoic acid-co-glycolic acid) [P(3HB-co-GL)]. The polyhydroxyalkanoic acid (PHA) for which the production method is provided by the present invention can be poly(3-hydroxybutanoic acid).

[0033] Culture media and conditions for PHA biosynthesis In the production method of the present invention, the microorganism is cultured in a medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and the microorganism can synthesize PHA using any one selected from the group consisting of alginic acid and alginic acid decomposition products as a carbon source. Alginic acid is a polysaccharide contained in brown algae and the like, and is a polymer in which two types of uronic acid (β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G)) are polymerized in a linear chain. The alginic acid used in the present invention is not particularly limited, and may have any degree of polymerization and any amount ratio of M and G (M / G ratio). The alginic acid of the present invention also includes alginates, alginic acid esters, etc. Examples of alginates include sodium alginate, potassium alginate, ammonium alginate, calcium alginate, etc. Examples of alginic acid decomposition products include partial decomposition products by enzymes and partial decomposition products by hydrolysis, such as uronic acid.

[0034] The concentration of alginic acid or alginic acid decomposition product in the culture medium for PHA biosynthesis can be appropriately set depending on the type of alginic acid or alginic acid decomposition product, the type of microorganism, etc., but is preferably in the range of 0.5% by mass to 20% by mass, more preferably in the range of 1% by mass to 10% by mass, and particularly preferably in the range of 1% by mass to 5% by mass. For example, in the case of sodium alginate, the range of 0.5% by mass to 20% by mass is preferably, more preferably in the range of 1% by mass to 10% by mass, even more preferably in the range of 1% by mass to 5% by mass, and particularly preferably in the range of 2% by mass to 4% by mass.

[0035] A nitrogen-limited inorganic salt medium can be used as a microorganism culture medium for PHA biosynthesis. The medium can contain an inorganic nitrogen source (e.g., ammonium sulfate, ammonium nitrate, urea) in addition to the carbon source described above. The medium can also contain minerals and trace elements, such as salts of phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), cobalt (Co), copper (Cu), zinc (Zn), and nickel (Ni). Specific examples of substances containing minerals and trace elements include calcium chloride, magnesium sulfate, potassium nitrate, sodium chloride, iron (III) chloride, calcium chloride, cobalt chloride, copper (II) sulfate, nickel (II) chloride, and chromium (III) chloride.

[0036] In the production method of the present invention, the culture for PHA biosynthesis can be carried out at a temperature in the range of about 20°C to 40°C and at a pH in the range of about 5 to 10. The culture temperature and pH can be appropriately adjusted according to the microorganism to be cultured. For example, among the above-mentioned microorganisms, 1) the microorganism deposited under accession number NITE P-02758 (strain 5-11-6-3 in the Example) and 2) the microorganism deposited under accession number NITE P-02759 (strain 5-28-6-1 in the Example) are microorganisms isolated by screening under acidic conditions. Therefore, the culture for PHA synthesis using the microorganism deposited under accession number NITE P-02758 or NITE P-02759 can be carried out in an acidic medium.

[0037] When marine bacteria are used as the microorganisms capable of alginate utilization and PHA synthesis, the concentration of sodium chloride in the culture medium can be set appropriately depending on the type of marine bacteria, etc., but it is preferably adjusted to a range of 0.5% by mass to 20% by mass, more preferably 1% by mass to 10% by mass, and particularly preferably 1% by mass to 8% by mass.

[0038] In the production method of the present invention, the culture for PHA synthesis is carried out under aerobic conditions, and an aeration stirring type culture tank or a bubble column type culture tank can be used. The size of the culture tank can be, for example, 150 milliliters or more. When culturing is carried out on an industrial scale, the size of the culture tank can be, for example, 100 liters or more. The culture can be carried out while continuously or intermittently supplying a carbon source or other nutrient sources. The carbon source concentration in the medium can be set in the range of about 0.5% by mass to about 5.0% by mass. The stirring speed can be, for example, 100 to 120 rpm. The number of days for culture can be set in the range of 1 to 7 days, but is not limited thereto. The culture can be carried out while controlling conditions such as temperature, pH, carbon source concentration, aeration amount, stirring speed, and culture time.

[0039] PHA Recovery PHA accumulated in the microbial cells can be collected by known methods (e.g., Patent Document 3). For example, the cells are separated from the culture using a separation means such as a centrifuge, and the cells are washed with distilled water, methanol, or the like, and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. Cell components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, the supernatant is removed by filtration or centrifugation, and the PHA can be collected by drying.

[0040] The production method of the present invention may include a previous step of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or acid. Extraction of alginic acid from seaweed can be performed by known methods (for example, JP 2017-21034 A). For example, alginic acid can be extracted from seaweed by the following method. In seaweed, alginic acid (AL-) is trapped as an inorganic ion salt with calcium ions, etc., so sodium carbonate is added to the seaweed and heated to extract sodium ions (Na + ) and calcium ions (Ca 2+ ) is subjected to ion exchange to extract alginic acid as water-soluble sodium alginate (alginic acid extraction). Next, calcium carbonate is removed from the alginic acid extract. The alginic acid extract from which calcium carbonate has been removed is acidified to precipitate alginic acid, or calcium ions are added to precipitate calcium alginate, which is then filtered to obtain alginic acid or calcium alginate.

[0041] The seaweed from which alginic acid is extracted is a macroalgae selected from brown algae, red algae, green algae, etc., and brown algae is particularly preferred. This is because brown algae are rich in alginic acid. Examples of brown algae include macroalgae belonging to the Laminariales and Fucales orders, such as kelp, wakame, and oenothera, but are not limited to these, and brown algae growing in the seas of the world can be used.

[0042] The alginic acid extracted as described above may be decomposed with an enzyme or an acid and then added to the medium as a carbon source. For the decomposition with an enzyme, alginate lyase (EC.4.2.2.3) or the like can be used. For the hydrolysis reaction with an acid, an inorganic acid such as hydrochloric acid or phosphoric acid, a sulfonic acid such as sulfuric acid, or a carboxylic acid such as acetic acid or formic acid can be used.

[0043] The present invention provides a method for producing a bioplastic product, which includes the steps of carrying out the above-mentioned production method to obtain polyhydroxyalkanoic acid (PHA) and obtaining a plastic product containing 25% by mass or more of the PHA obtained in the previous step. The Japan Bioplastics Association states that a product must have a bioplastic content of 25% by weight or more in order to meet the criteria for identifying and labeling biomass plastics. The bioplastic content is the ratio of the mass of biomass plastic components in a biomass plastic product to the total mass.

[0044] The PHA produced by the production method of the present invention can be used as a raw material for various plastic products. Examples of plastic products containing PHA include films and sheets that can be used as packaging for frozen foods and cushioning materials, stationery such as ballpoint pens, disposable tableware such as straws, forks, spoons and plates, civil engineering and construction materials, multipurpose films for agricultural and marine materials, seedling pots, fishing lines, fishing nets, etc.

[0045] microorganisms Genus Covetia The present inventors have found that several microbial strains isolated from seaweed washed up on Goishi Beach in Ofunato Bay, Ofunato City, Iwate Prefecture, have the ability to assimilate alginic acid and synthesize PHA. In particular, the microbial strains 5-28-6-1 and 5-11-6-3 described in the Examples are bacilli, gram-negative (FIG. 8), and grow in a medium with a NaCl concentration of about 5%. The microbial strains 5-28-6-1 and 5-11-6-3 each have a 16S rRNA gene containing the base sequence set forth in SEQ ID NO: 1 or 2. Analysis of a sequence database using the homology search program BLAST (Altschul SF et al., 1990 Basic local alignment search tool. J. Mol. Biol. Vol. 215, pp. 403-410) based on the base sequence of the 16S rRNA gene suggests that they may belong to the genus Covetia.

[0046] Covetia bacteria are aerobic, slightly halophilic (0.5-20%), and have been reported to grow at temperatures between 4 and 42°C and pH between 4 and 11 (Romanenko LA et al., International Journal of Systematic and Evolutionary Microbiology, 63, 288-297, (2013)). They have also been reported to accumulate P(3HB) intracellularly using glucose as a carbon source (Romanenko LA et al. 2013; Arahal DR et al., Appl. Microbiol. 25, 207-211, 2002), but there have been no reports of PHA accumulation using alginate as a carbon source prior to this application. Covetia bacteria have also been reported to have round, shiny, smooth, and cream-colored colonies (Romanenko LA et al. 2013). The colonies of 5-28-6-1 and 5-11-6-3 strains observed during single bacterial isolation were also round, shiny, smooth, and cream-colored, matching the colony morphology reported for the above-mentioned Covetia bacteria. In addition, the conditions under which 5-28-6-1 and 5-11-6-3 strains can grow also matched those reported for the above-mentioned Covetia bacteria. Therefore, it was suggested that 5-28-6-1 and 5-11-6-3 strains may belong to the Covetia genus.

[0047] The phenotypic characteristics of the 5-11-6-3 strain were compared with those of the DSM4741 strain (Arahal DR et al., 2002, supra), which is the type strain of Cobetia marina. As a result, it was found that while Cobetia marina can grow in a medium with a NaCl concentration of 0%, the 5-11-6-3 strain cannot grow in a medium with a NaCl concentration of 0%. In addition, the assimilation of L-arabinose and D-mannose is different (Table 22 in Example 6). Therefore, the 5-11-6-3 strain is a Cobetia bacterium, and although it is closely related to Cobetia marina, it may be a different species.

[0048] The present invention provides a bacterium of the genus Covetia having the ability to utilize alginic acid and synthesize PHA. The present invention provides a Covetia bacterium having a 16S rRNA gene comprising the following base sequence (a) or (b): (a) a nucleotide sequence as set forth in SEQ ID NO: 1 or 2; (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0049] The present invention provides a bacterium of the genus Covetia having a 16S rRNA gene containing the following base sequence (a) or (b) and having the ability to utilize alginate and synthesize PHA: (a) a nucleotide sequence as set forth in SEQ ID NO: 1 or 2; (b) A nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

[0050] The nucleotide sequence and nucleotide sequence identity can be analyzed by the methods described above. Whether or not a bacterium has the ability to utilize alginic acid and / or synthesize PHA can be determined by the methods described above. The Covetia bacterium of the present invention has the ability to utilize alginic acid and synthesize PHA, and therefore can be used in a method for producing bioplastics using seaweed as a raw material.

[0051] microorganism deposit The 5-28-6-1 strain has been deposited with the depository institution by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture) as follows. The 16S rRNA gene of the 5-28-6-1 strain has the base sequence shown in column number 1. (i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Depository Center (NITE-NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (ii) Trust date (deposit date): August 8, 2018 (iii) Accession number NITE P-02759 (Cobetia sp. IU180733JP01 (5-28-6-1) strain)

[0052] The 5-11-6-3 strain has been deposited at the depository institution by Iwate University (Address: 3-18-8 Ueda, Morioka City, Iwate Prefecture) as follows. The 16S rRNA gene of the 5-11-6-3 strain has the base sequence shown in SEQ ID NO:2. (i) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Depository Center (NITE-NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (ii) Trust date (deposit date): August 8, 2018 (iii) Accession number NITE P-02758 (Cobetia sp. IU180733JP01 (5-11-6-3) strain)

[0053] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" is based on mass, and numerical ranges are described as including their endpoints.

[0054] Reagents and Equipment The reagents and devices used in the examples are as follows. [Table 1]

[0055] [Table 2]

[0056] [Table 3]

[0057] [Table 4]

[0058] [Table 5]

[0059] [Table 6] EXAMPLES

[0060] Example 1 Isolation of bacteria from natural samples A total of 31 natural samples were collected from the vicinity of Goishi Beach in Ofunato City, Iwate Prefecture on June 20, 2017, and were stored at 4°C before being used in the experiment. The natural samples included seawater, seaweed, shells, and sea slugs.

[0061] The collected natural samples were cultured for 2 to 3 days (scale: 5 mL test tube, temperature: 30°C, stirring speed: 111 rpm, medium: ZoBell 2216E seawater medium, pH: 5, 7 or 9). By using seawater from Ofunato City, Iwate Prefecture as the medium, the initial culture was performed under conditions close to the environment in which the samples were collected. HCl and NaOH were used to adjust the pH. The seawater for the medium was filtered through filter paper (No. 2.110 mm) before use. 5 mL of the prepared medium was dispensed per test tube and autoclaved (121°C, 15 min).

[0062] The seaweed samples were cut into pieces of approximately 1cm x 1cm size and added directly to the medium. Solid samples (shells, sea slugs) were suspended in 5mL of 0.1M potassium phosphate buffer (KPB, pH7.0) and 200μL of the suspension was added to the medium. Shells were crushed and the contents ground in a mortar and pestle and suspended in KPB, while sea slugs were ground in a mortar and the resulting liquid was suspended in KPB. 200μL of liquid samples (seawater) were added directly to the medium.

[0063] On the first day of culture, regardless of pH, the medium, which was transparent immediately after inoculation, was observed to have turned white or yellow and cloudy in all samples. Single bacteria were isolated using culture fluid from the second to third day. Figure 2A shows the state of the culture of some samples.

[0064] In order to obtain a single colony from the medium cultured with the natural sample, single bacterial isolation was performed using the medium shown in Table 7 at a culture temperature of 30°C for 3 days. The medium cultured with the natural sample was diluted and inoculated onto the medium shown in Table 7. The natural sample was diluted by taking 10 μL of the culture liquid and mixing it with 990 μL of sterilized RO water. This procedure was repeated for 10 4 ,10 6 ,10 8 The plates were then diluted 2x and 200 μL of each solution was inoculated. Observations were performed daily, and plates with sufficient growth were stored at 4°C until the next experiment.

[0065] [Table 7]

[0066] result On one agar plate, multiple colonies of different colors, morphologies, and sizes were confirmed (Figure 2B). These were visually selected, and a total of 311 strains were obtained (Table 8). There was no notable trend in the number of colonies at each pH level.

[0067] [Table 8]

[0068] Example 2 Selection of PHA synthesis candidate strains using Nile Red (primary screening) In this example, as the primary screening, the strain obtained by the single-cell isolation in Example 1 was cultured in a medium containing Nile Red to select a candidate strain for PHA synthesis. Nile Red is a hydrophobic dye that reacts with neutral lipids in cells to turn red, making it possible to visually obtain candidate strains. Two types of medium, one containing Nile Red and one not, were prepared, and the color change was observed with the naked eye by inoculating both plates. The colonies that turned red were stored at 4°C until the next experiment.

[0069] The composition and culture conditions of the medium containing Nile Red are shown in Tables 9 and 10. The medium was autoclaved (121°C, 15 min), Trace Element was filter sterilized (Minisart (registered trademark)-RC25, sartorius, RC, pore size 0.45 μm), and sodium alginate was autoclaved (121°C, 15 min) separately from the medium. Nile Red was dissolved in dimethyl sulfoxide and used without sterilization. The final concentration of sodium alginate in the medium was 1%. Colonies with different colors and morphologies were selected from the ZoBell 2216E seawater plate, picked with a toothpick, and inoculated onto two plates. The plates were cultured at 30°C for 5 days, and the Nile Red-containing plate was cultured in the dark.

[0070] [Table 9]

[0071] [Table 10]

[0072] result The plates were observed with the naked eye every day, and red-colored strains were selected, yielding a total of 171 strains (Table 11). After the culture was completed, the plates were stored at 4°C. [Table 11]

[0073] Example 3 Selection by gas chromatography-mass spectrometry (GC-MS) method (secondary screening) In this example, the selection was performed by GC-MS method as the secondary screening. Since Nile Red reacts with substances other than PHA and turns red, the strain selected in the primary screening was cultured, PHA polymer was extracted, and the extract was ethanolyzed, and the PHA monomer was detected by GC-MS method for further selection. [ka]

[0074] (Culture) The PHA synthesis candidate strains obtained in the primary screening were cultured in test tubes (10 mL). The medium shown in Table 12 was autoclaved (121°C, 15 min), and the Trace Element (Table 11) and sodium alginate were added to the medium after sterilization separately in the same manner as above. Colonies of the PHA synthesis candidate strains obtained in the primary screening were separated with a toothpick, and the toothpick was placed in the medium and cultured with shaking. Sodium alginate was used as a carbon source, with a final concentration of 1%. The culture was performed at a temperature of 30°C and an agitation speed of 111 rpm for 72 hours, and the culture time was further extended for samples with slow growth.

[0075] [Table 12]

[0076] Of the 171 strains obtained in the primary screening, 121 samples could be cultured. Some of the culture results are shown in Figure 3. PHA was extracted from these samples and subjected to GC-MS analysis.

[0077] (PHA extraction) After the cultivation, the pH and OD of the culture solution were 660 The bacterial mass was measured, and the cells were collected by suspension in RO water twice and centrifugation three times. Centrifugation was performed at 8,490×g or 7.670×g. The collected cells were pre-frozen at -30°C. The tube containing the cells was then covered with parafilm, a hole was made in the film, and the cells were freeze-dried for at least 24 hours using a freeze-dryer. After freeze-drying, the dried cells were weighed and crushed finely, then transferred to a screw-top test tube, and 5 mL of chloroform was added per 200-300 mg of cells. After vortexing, the cells were heated at 70°C for two days. Vortexing was performed as necessary. After heating, the cells were removed by passing through a filter, and the filtrate was heated in a draft to dryness. The weight of the extract was determined by measuring the weight of the screw-top test tube before and after filtration. The dried sample was stored at room temperature.

[0078] (Preparation of GC-MS sample) 1 mL of chloroform was added to the extract, and then it was dissolved by heating. 3.4 mL of 99.5% ethanol and 400 μL of HCl (stock solution) were added, and the mixture was stirred by vortexing for 1 minute and heated at 100°C for 4 hours using a heat block (ethanolysis treatment) (Y. Arai, et al., Plant Cell Physiol. 43(5), 555-562, (2002)). During heating, the mixture was vortexed for 1 minute every 30 minutes. The mixture was cooled once on ice, and 4 mL of a 0.65 M NaOH + 0.9 M NaCl mixture and 2 mL of 0.25 M Na2HPO4 were added, vortexed for 1 minute, and the chloroform layer was confirmed to be neutral using pH test paper. The solution was then centrifuged at 800 × g for 5 minutes, and the chloroform layer was collected at the bottom. The chloroform layer was passed through a column made by packing glass wool and Na2SO4 in a Pasteur pipette, and the chloroform layer was transferred to a screw-top test tube containing molecular sieves that had been preheated at 180°C for 2 hours to dehydrate it. This was used as a sample and was stored at -30°C until analysis.

[0079] To measure the amount of synthesized PHA, P(3HB), one of the most common PHAs, was subjected to ethanolysis and a calibration curve was created using 3HB ethyl as the standard substance. To create the calibration curve, P(3HB) was diluted with chloroform to prepare concentrations of 10, 50, 100, 500, and 1000 μg / mL, and each of these was used as a sample subjected to ethanolysis.

[0080] (GC-MS method conditions) An HP-5 column (length 30 m, film thickness 0.25 μm, inner diameter 0.25 mm) was used. The analytical conditions are as shown in Tables 13 and 14. [Table 13]

[0081] [Table 14]

[0082] The mass spectrum peak of the 3HB ethyl standard substance was confirmed at a retention time of around 4.7. As a result of analyzing 121 samples, a peak showing the mass spectrum of 3HB ethyl, which is thought to be derived from P(3HB), was detected in multiple samples. 14 samples that showed a peak at a retention time of 4.76 to 5.00 were selected as candidate strains for PHA synthesis (Table 15). Among the 14 samples that showed a peak at a retention time of 4.76 to 5.00, the analysis results of the 5-28-6-1 strain and the 5-11-6-3 strain are shown in Figures 4A and B.

[0083] The amount of P(3HB) was calculated from a calibration curve using 3HB ethyl as a standard substance, and the P(3HB) accumulation rate was calculated by dividing the amount of P(3HB) by the dry cell weight (Table 15). [Table 15]

[0084] In Examples 1 and 2, 31 samples of seaweed, etc., sampled from the natural world were cultured in seawater medium and single-cell isolation was performed to obtain a total of 311 strains. After that, selection was performed using a medium containing Nile Red to obtain 171 PHA synthesis candidate strains. These strains were cultured in a PHA synthesis medium, and after polymer extraction, samples subjected to ethanolysis treatment were detected by GC-MS analysis to further narrow down the candidate strains. As a result, 3HB ethyl, an ethanolysis product of P(3HB), one of the most major PHAs, was detected in 14 strains.

[0085] In GC-MS measurements, the sample is first converted into a monomer, so it is not possible to determine whether a polymer has been synthesized. 1 It is necessary to perform H-NMR analysis. 1 A certain amount of analytical sample is required for H-NMR analysis. Therefore, in the following Example 4, polymer synthesis was performed by culturing on a multiple test tube and Sakaguchi flask scale, 1 It was decided to confirm the synthesis of PHA by H-NMR analysis.

[0086] Example 4 1 H-Nuclear Magnetic Resonance ( 1 H-NMR) analysis 1. Cultivation at Sakaguchi flask scale Scale-up culture was carried out in a Sakaguchi flask for the 14 strains for which PHA synthesis was suggested by the GC-MS method in Example 2. Seed culture was carried out in a test tube at a 10 mL scale as described in Example 2, and main culture was carried out by subculturing 3 mL of the seed culture solution. Culture at the Sakaguchi flask scale was carried out at a temperature of 30°C and an agitation speed of 111 rpm for 24 to 72 hours, and the culture time was further extended for samples with slow growth. At this time, the culture time of the 9-15-0-1 strain was extended because it grew slowly.

[0087] The results of the main culture are shown in the table below. [Table 16]

[0088] As shown in the table above, the relationship between dry cell weight and extract weight over time was clarified, and in most samples, the dry cell weight and extract weight reached their peak in the 1-2 day culture, and tended to decrease in the 3 day culture. The reason for this is thought to be that most strains grow quickly, and nutrients may have become depleted around the 1-2 day culture. The fact that they grow quickly and take a short time to synthesize PHA is thought to be an advantage when considering industrial use.

[0089] 2. Polymer Purification and Sample Preparation A chloroform extract was obtained from the above culture by the PHA extraction method described in Example 3. This was dissolved in a small amount of chloroform, and an excess amount (10 times or more) of hexane was added thereto to precipitate the polymer, followed by purification of the polymer. The purified polymer was dissolved by adding 2 mL of chloroform-D (D, 99.8%) + 1 v / v% TMS and heating.

[0090] 3.1 H-NMR analysis As a result of the analysis, peaks specific to P(3HB) homopolymer, which is one of PHAs, were observed in 13 of the 14 strains (1.26-1.28, 2.44-2.64, 5.24-5.28 ppm), and no peaks were detected for the monomer 3HB. Figures 5A and 5B show part of the analysis results (5-28-6-1 strain and 5-11-6-3 strain). The 3HB peaks were observed at 1.20-1.21, 2.30-2.43, and 4.13-4.19 ppm, so P(3HB) homopolymer can be distinguished from 3HB. The peak detected near 1.5 ppm is thought to be residual water, and the peak near 7.26 ppm is thought to be deuterated chloroform. The peak specific to P(3HB) homopolymer was not detected in the 9-10-8-2 strain.

[0091] After culture in test tubes 1 H-NMR analysis confirmed that 13 strains were P(3HB) homopolymers, suggesting that the strains may convert alginate to acetyl-CoA and synthesize P(3HB) through the previously reported three-step enzymatic reaction (Sudesh K. et al, Prog. Polym. Sci. 25 (2000) 1503-1555; Hiroyuki Takeda et al., Energy Environ. Sci.,2011, 4, 2575).

[0092] Example 5 Species identification: Determination of 16S rRNA base sequence Nucleic acid was extracted from the bacterial cells, and after ethanol precipitation, the nucleic acid concentration was measured and agarose electrophoresis was performed. DNA extraction was confirmed in all samples, so the samples were subjected to the next procedure. PCR amplification of 16S rDNA or 18S rDNA was performed on the DNA extracts. Since it was not clear whether this strain was a bacterium or a fungus, both 16S rRNA primers and 18S rRNA primers were used. [Table 17]

[0093] PCR was performed using TaKaRa Ex Taq with the following reaction mixture composition: 0.25 μL of TaKaRa Ex Taq (5 U / μL); 5 μL of 10×Ex Taq Buffer; 4 μL of dNTP mixture (each 2.5 mM); 3 μL of primer (10 μM); 3-5 μL of template (<500 ng); up to 50 μL of sterile MQ water). The reaction conditions were 98°C for 10 sec, 55°C for 30 sec, and 72°C for 90 sec for 30 cycles. As a result, a band of approximately 1.5 kbp corresponding to 16S rRNA was confirmed in all samples except for 5-10-6-1.

[0094] Subsequent experiments were carried out using strains 5-28-6-1 and 5-11-6-3, which showed stable and high P(3HB) accumulation rates. The 16S rRNA of the 5-28-6-1 and 5-11-6-3 strains was amplified by PCR and inserted into the pTA2 vector, which was then transformed into E. coli JM109. Recombinants were randomly selected, and after 16 hours of cultivation, the cells were harvested and the plasmids were extracted.

[0095] The obtained plasmid DNA was subjected to sequence analysis, and the base sequence of 1496 bp, which is the full length of the region flanked by the above primers for amplifying the 16S rRNA gene, was determined for both strains 5-28-6-1 and 5-11-6-3. A homology search was performed using the BLAST program. As a result, strains 5-28-6-1 and 5-11-6-3 showed 99% homology with the 16S rRNA of Cobetia marina. The base sequences of the 16S rRNA strongly suggested that the microorganisms of strains 5-28-6-1 and 5-11-6-3 were likely to be Cobetia bacteria (Table 18). There were four base differences between strains 5-28-6-1 and 5-11-6-3 (Figure 7).

[0096] [Table 18]

[0097] Example 6 Species identification: phenotypic characteristics of strain 5-11-6-3 The characteristics of the bacterium strain 5-11-6-3, which was suggested to belong to the genus Covetia based on the 16S rRNA sequence analysis, were analyzed. Motility tests were performed by inoculating the bacteria into test tubes containing agar medium with a NaCl concentration of 2%, a modified version of the medium composition of Rudolph Hugh et al. (The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria, J Bacteriol. 1953 Jul;66(1):24-26.), and observing the motility under a microscope. Motility tests were performed using sodium alginate and mannitol as carbon sources, and the bacteria were not observed to spread over the entire medium, but rather to grow along the puncture line. It is believed that the 5-11-6-3 strain does not have motility on agar.

[0098] To study the temperature range in which the bacteria can grow, a culture experiment was carried out using SW-5 medium (the composition of which is shown in Table 19). 30% Marine salts (the composition of which is shown in Table 20) was sterilized by filter (Minisart (registered trademark)-RC25, sartorius, RC, pore size 0.45 μm) and added to the medium to a final concentration of 5%. A certain amount of culture of 5-11-6-3 strain bacteria was added to SW-5 medium and cultured at temperatures of 4, 15, 25, 37, 42, or 45 ° C. The growth of the bacteria was confirmed in the range of 4 to 42 ° C. The bacteria was cultured for one week at 45 ° C, but the growth of 5-11-6-3 strain bacteria could not be confirmed.

[0099] [Table 19] [Table 20]

[0100] To investigate the range of NaCl concentrations that allows growth, a culture experiment was carried out in SW medium supplemented with 0-25% marine salts. A certain amount of the bacterial strain culture was added to SW-5 medium, and the growth status was confirmed after one week of culture at 37°C with NaCl concentrations of 0, 0.5, 3, 5, 10, 15, 20, or 25%. Growth of the 5-11-6-3 strain bacteria was confirmed at 0.5-20% marine salts. However, no growth was confirmed at 0%.

[0101] Cytochrome oxidase test filter paper (Nissui Pharmaceutical) was used for the oxidase test. 5-11-6-3 strain bacteria were cultured in fresh PHA synthetic medium for 4 days. The test paper was placed in a petri dish, a few drops of MQ water were dripped onto it to moisten the entire filter paper, and the cultured bacteria on solid medium was immediately applied onto it with a platinum loop. If the applied area turned deep blue within one minute, it was judged to be a positive bacterium. The color of the test paper did not change even after one minute had passed. Therefore, this strain is considered to be oxidase negative.

[0102] Nutritional tests were performed using the medium whose composition is shown in Table 21. The carbon sources used were L-arabinose, D-glucose, glycerol, myo-inositol, D-mannitol, D-mannose, D-sorbitol, sucrose, and lactose. The carbon source concentration was adjusted to 10% and filter sterilized before use. Growth of the 5-11-6-3 strain bacteria was confirmed using carbon sources other than L-arabinose, D-sorbitol, and lactose (D-glucose, glycerol, myo-inositol, D-mannitol, D-mannose, and sucrose).

[0103] [Table 21]

[0104] The results of analyzing the properties of the 5-11-6-3 strain bacterium are shown in Table 22. The phenotypic properties of the previously reported Cobetia genus bacterium (Cobetia marina DSM4741 strain (type strain)) are also shown in Table 22 (Arahal DR et al., 2002, supra).

[0105] [Table 22]

[0106] The genus Cobetia is a gram-negative bacterium, and the 5-11-6-3 strain is also a gram-negative bacterium, so the characteristics matched those of the 5-11-6-3 strain. In addition, the growth temperature and oxidase negativity of the 5-11-6-3 strain were also consistent with those of the 5-11-6-3 strain. However, in terms of the NaCl concentration at which they can grow, Cobetia marina can grow in a medium with a NaCl concentration of 0%, while the 5-11-6-3 strain could not grow in a medium with a NaCl concentration of 0%. Furthermore, in a nutritional test, the types of sugars used for growth were different between the 5-11-6-3 strain and Cobetia marina. Therefore, it was suggested that this strain is a Cobetia bacterium, but may not be Cobetia marina.

[0107] Example 7: Examination of culture conditions (sodium alginate concentration) A colony of the bacteria of the genus Covetia, strain 5-11-6-3, was seed-cultured for 2 days on a PHA synthetic plate medium (medium described in Table 12, pH 5.0). After 2 days of culture, 3 mL of the seed culture was inoculated and main culture was performed in triplicate. To examine the effect of sodium alginate concentration on PHA synthesis, the main culture medium had an NaCl concentration of 2% and a sodium alginate concentration of 1%, 2% or 3%, and the medium was cultured for 12, 24 or 36 hours. After main culture, the pH and OD 660After measuring the amount of the bacteria, the mixture was transferred to a centrifuge tube, centrifuged at 7,670×g for 15 minutes, and the supernatant was discarded by decantation. The bacteria were suspended in RO water and centrifuged again under the same conditions, followed by decantation. The bacteria were suspended in RO water and centrifuged again, and finally the supernatant was discarded and the bacteria were stored at -30°C. The pre-frozen bacteria were freeze-dried for at least 24 hours using a freeze dryer (EYELA FDS-1000, Tokyo Rikakikai Co., Ltd.). They were then crushed in a mortar and the weight was measured. Approximately 5 mL of chloroform was added per 200-300 mg of dried bacteria, and extraction was performed at 70°C for 48 hours using a heat block. After completion of extraction, the bacteria were filtered using a filter (DISMIC (registered trademark)-25HP, Advantec, PTFE, pore size 45μm), and the extract was dried by heating at 55°C using a heat block. If the extract was dirty, it was dissolved in a small amount of chloroform, and then an excess amount (approximately 10 times or more) of hexane was added to precipitate P(3HB), after which the hexane was removed and the mixture was dried. The weight of the precipitated P(3HB) was calculated from the weight of the test tube before and after the experiment, and the accumulation rate of P(3HB) was calculated from the weight of the dried cells. The results are shown in Table 23. [Table 23]

[0108] The results showed that the addition of 3% sodium alginate to the medium resulted in the highest bacterial growth and P(3HB) accumulation. The P(3HB) accumulation rate reached 46.6% after 36 hours, and the amount of P(3HB) synthesis reached a maximum of 297.5 mg after 24 hours of culture. [Industrial Applicability]

[0109] The present invention is useful in the plastics industry. [Sequence List Free Text]

[0110] SEQ ID NO: 1: 16S rRNA gene sequence of the microbial strain 5-28-6-1 SEQ ID NO: 2: 16S rRNA gene sequence of the microbial strain 5-11-6-3 SEQ ID NO: 3: Primer for amplifying 16S rRNA gene SEQ ID NO: 4: Primer for amplifying 16S rRNA gene SEQ ID NO: 5: Primer for amplifying 18S rRNA gene SEQ ID NO: 6: Primer for amplifying 18S rRNA gene

[0111] SEQUENCE LISTING <110> Iwate University <120> A method for generating Polyhydroxyalkanoates from Alginate <130> 195139K <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 1496 <212> DNA <213> Cobetia sp 5-28-6-1 <400> 1 attgaacgct ggcggcaggc ttaacacatg caagtcgagc ggaaacgatt ctagcttgct 60 agaaggcgtc gagcggcgga cgggtgagta atgcatggga atctgcccga tagtggggga 120 caacctgggg aaactcaggc taataccgca tacgtcctac gggagaaagc aggggatctt 180 cggaccttgc gctatcggat gagcccatgt cggattagct tgttggtgag gtaacggctc 240 accaaggcga cgatccgtag ctggtctgag aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaggcc ttcgggttgt aaagcacttt cagcgagga gacgcttcg ggattaatac tcccgagga agacatcact cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc 540 gtaaagcgcg cgtaggtggc tagtcagcc aggtgtgaaa gccccgggct caacctggga acggcatctg gaactgcttg gctagagtgc aggagagga ggtagaattc ccggtgtagc 660 ggtgaaatgc gtagagatcg ggaggaatac cagtggcga ggcggccttc tggactgaca 720 ctgacactga ggtgcgaaag cgtgggtagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttgggtc ccttgaggac ttagtggcgc agctaacgca 840 ataagttgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctacc 960 cttgacatcc agaggacttt ccagagatgg attggtgcct tcgggaactc tgagacaggt 1020 gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg taacgagcgc 1080 aacccctatc cttatttgcc agcgagtaat gtcgggaact ctaaggagac tgccggtgac 1140 aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacgggt agggctacac 1200 acgtgctaca atggcaagta caaagggttg caatacggcg acgtggagcc aatcccataa 1260 agcttgcctc agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt 1320 aatcgtgggt cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440. 1440 cggtgtggtt catgactggg gtgaagtcgt aacaaggtag ccctagggga acctgg <210> 2 <211> 1496 <212> DNA <213> Cobetia sp 5-11-6-3 <400> 2 attgaacgct ggcggcaggc ttaacacaag caagtcgagc ggaaacgatt ctagcttgct agaaggcgtc gagcggcgga cgggtgagta atgcatggga atctgcccga taatggggga caacctgggg aaactcaggc fathercgca tacgtcctac gggagaaagc aggggatctt cggaccttgc gctatcggat gagcccatgt cggattagct tgttggtgag gtaacggctc 240 accaaggcga cgatccgtag ctggtctgag aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaggcc ttcgggttgt aaagcacttt cagcgagga gaacgcttcg ggattaac tcccgaggaa agacatcact cgcagaagaa gcaccggcta 480 actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc 540 gtaaagcgcg cgtaggtggc taagtcagcc aggtgtgaaa gccccgggct siacctggga 600 acggcatctg ggactgcttg gctagagtgc aggagaa ggtagaattc ccggtgtagc 660 ggtgaaatgc gtagagatcg ggaggaatac cagtggcgaa ggcggccttc tggactgaca 720 ctgacactga ggtgcgaaag cgtgggtagc aacaggatt agataccctg gtagtccacg 780 ccgtaaacga tgtcaacag ccgttgggtc ccttgaggac ttagtggcgc agctaacgca 840 ataagttgac cgcctgggga gtacggccgc aaggttaaaa ctcaatgaa ttgacgggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagac cttacctacc 960 cttgacatcc agaggacttt ccagagatgg attggtgcct tcgggactc tgagacaggt 1020 gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg taacgagcgc 1080 aacccctatc cttatttgcc agcgagtaat gtcgggaact ctaaggagac tgccggtgac 1140 aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacgggt agggctacac 1200 acgtgctaca atggcaagta caaagggttg caatacggcg acgtggagcc aatcccataa 1260 agcttgcctc agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt 1320 aatcgtggat cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 caccatggga gtggactgca ccagaagtgg ttagcctaac cttcgggagg gcgatcacca 1440 cggtgtggtt catgactggg gtgaagtcgt aacaaggtag ccctagggga acctgg 1496 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> primer <400> 3 agagtttgat cctggctcag 20 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <220> <223> primer <400> 4 aaaggaggtg atccagcc 18 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> primer <400> 5 atctggttga tcctgccagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> primer <400> 6 aatgatcctt ccgcaggttc 20

Claims

1. Microorganisms deposited under accession numbers NITE P-02758 or NITE P-02759.

2. A method for producing polyhydroxyalkanoic acid (PHA) using alginic acid as a carbon source, comprising culturing a Covetia bacterium having the ability to utilize alginic acid and synthesize PHA in a medium containing any one selected from the group consisting of alginic acid and alginic acid decomposition products, and extracting PHA from the culture, wherein the bacterium is the microorganism described in claim 1.

3. The method according to claim 2, further comprising the steps of extracting alginic acid from seaweed and / or decomposing the alginic acid extracted from seaweed with an enzyme or an acid as a pre-step.

4. The method according to claim 2 or 3, wherein the bacterium is a genetically modified bacterium.

5. The method according to any one of claims 2 to 4, wherein the concentration of alginic acid or alginic acid decomposition product in the medium is in the range of 0.5% by mass to 20% by mass.

6. The method according to any one of claims 2 to 5, wherein the concentration of sodium chloride in the medium is in the range of 0.5% by mass to 20% by mass.

7. The method according to any one of claims 2 to 6, wherein the PHA comprises 3-hydroxybutanoic acid monomer units.

8. The method according to claim 7, wherein the PHA is poly(3-hydroxybutanoic acid).

9. Carrying out the method according to any one of claims 2 to 8 to obtain polyhydroxyalkanoic acid (PHA); A method for producing a bioplastic product, comprising the step of obtaining a plastic product containing 25% by mass or more of the PHA obtained in the above step.

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