WO2020174987A1 - Transgenic microorganism, and method for producing polyhydroxyalkanoic acid - Google Patents

Transgenic microorganism, and method for producing polyhydroxyalkanoic acid Download PDF

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Publication number
WO2020174987A1
WO2020174987A1 PCT/JP2020/003155 JP2020003155W WO2020174987A1 WO 2020174987 A1 WO2020174987 A1 WO 2020174987A1 JP 2020003155 W JP2020003155 W JP 2020003155W WO 2020174987 A1 WO2020174987 A1 WO 2020174987A1
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gene
acid
polyhydroxyalkanoic acid
producing
genus
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PCT/JP2020/003155
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French (fr)
Japanese (ja)
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尚志 有川
俊輔 佐藤
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株式会社カネカ
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Priority to JP2021501777A priority Critical patent/JP7425783B2/en
Publication of WO2020174987A1 publication Critical patent/WO2020174987A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.
  • microorganisms Against the backdrop of environmental problems, food problems, increased awareness of health and safety, and increased awareness of nature or nature, the significance and importance of substance production using microorganisms (fermentation production, bioconversion, etc.) is increasing.
  • the production of substances by microorganisms is also applied to the production of protein drugs and nucleic acids for gene therapy.
  • the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria are actively being applied industrially.
  • ⁇ 18 is a thermoplastic polyester that is produced and accumulated in cells of many microbial species as an energy storage substance and has biodegradability.
  • non-petroleum-derived plastics are attracting attention due to increasing environmental awareness.
  • microorganisms are produced and accumulated in the fungus body!...8 because they are incorporated into the carbon cycle process in the natural world. It is expected that the adverse effect on the product will be small, and its practical application is highly desired.
  • _ was eight production, for example, sugars, vegetable oil fat or fatty acid given as a carbon source in Cupriavidus bacterium, a child produces 1-1 eight by to accumulate 1 to 1 (viii) in the intracellular Are known (see Non-Patent Documents 2 and 3).
  • Non-Patent Document 4 reports that disruption of the gene II 31 encoding the fasin protein in a bacterium belonging to the genus Capriavidus accumulated a larger particle size of 1 to 1 than in the case of non-disruption. Therefore, it is possible to adopt this as a method to improve the separation and recovery efficiency of the target product. But,
  • the 1-disrupted strain was shown to significantly reduce the amount of PHA accumulated, and was not suitable for industrial production.
  • Non-Patent Document 1 8 ⁇ ⁇ 16 "30 ⁇ 8" ., a ., 1 ⁇ 1:. ⁇ . ⁇ 1
  • Non-Patent Document 2 38 I ⁇ 3., ⁇ 8 and ”6 I ⁇ 5 ⁇ I .6 I ⁇ 6 doors., 1 20 (3), 246-25 1 (201 5)
  • 1 to 1 is accumulated in the particulate in the microbial cells.
  • the cells are crushed to extract the particles, and the particles are separated from other cell components and collected.
  • the method of separation and recovery can be roughly classified into an organic solvent-based method and an aqueous method, but the use of an organic solvent has a high environmental load and is costly, and therefore the aqueous method is industrially preferable.
  • ! ⁇ 1 particles can be separated from the cell lysate containing !8 particles using a centrifuge or a separation membrane. ⁇ 02020/174987 3 ((171?2020/003155
  • the efficiency of separation and recovery depends on the size of 1 to 1 particles. That is, the larger the 1 to 1 particle accumulated in the microbial cell, the easier the separation and recovery using a centrifugal separator or a separation membrane, which leads to a reduction in production cost.
  • the present invention has It is an object of the present invention to provide a transformed microorganism capable of accumulating a bacterium, and a method for producing 1 to 1 using the transformed microorganism.
  • the present inventors have found that enhancement of the expression of the 8 2 365 gene (for example, the gene encoding the amino acid sequence set forth in SEQ ID NO: 1) whose function is unknown in the bacteria of the genus Capriavidus
  • the inventors have found that the particle size of 1 to 1 accumulated in microbial cells can be increased while maintaining the industrially desirable accumulation amount, and the present invention has been completed.
  • the present invention has a polyhydroxyalkanoic acid synthase gene
  • the present invention relates to a transformed microorganism having enhanced expression of 2365 gene.
  • the transformed microorganisms preferably belong to the genus Capriavidas, and more preferably, the transformed microorganisms of Capriavidus-neca laurel.
  • the present invention also relates to a method for producing polyhydroxyalkanoic acid, which comprises a step of culturing the transformed microorganism in the presence of a carbon source.
  • the carbon source may contain fats or oils or fatty acids, may contain sugar, and may contain carbon dioxide.
  • the polyhydroxyalkanoic acid is preferably a copolymer of two or more kinds of hydroxyalkane acids, more preferably a copolymer containing 3-hydroxyhexanoic acid as a monomer unit, and 3-hydroxy More preferably, it is a copolymer of butyric acid and 3-hydroxyhexanoic acid.
  • [001 1] According to the present invention, it is possible to provide a transformed microorganism capable of accumulating a large number of particles!, and a method for producing !! using the transformed microorganism. Book According to the invention, since PHA particles having a large particle size are accumulated in the microbial cells, it becomes easy to separate and recover PHA from the cell components after cell disruption, and it is possible to reduce the production cost.
  • the transformed microorganism according to the present invention is a transformed microorganism having a PHA synthase gene and enhanced expression of the A2365 gene.
  • the transformed microorganism according to the present invention has a PHA synthase gene, and is a microorganism transformed so that the expression of the A 2365 gene is enhanced (hereinafter, also referred to as A 2365 gene expression-enhanced strain). ..
  • the host of the transformed microorganism of the present invention is not particularly limited as long as it is a microorganism having a PHA synthase gene.
  • the bacterium include Ralstonia genus, Cupriavidus genus, W autersia genus, Aeromonas (Ae r omo nas) genus, Escherichia genus, Alcaligenes (A lcaligenes).
  • Preferable examples include bacteria belonging to the genus Pseud omo nas and the like. From the viewpoints of safety and PHA productivity, bacteria more preferably belonging to the genus Ralstonia, the genus Capriavidus, the genus Aeromonas, and the genus Woutercia are more preferred, and the bacteria belonging to the genus Capriavidus or the genus Aeromonas are still more preferred, and still more preferably capriavidus. It is a microorganism belonging to the genus, and particularly preferably Capriavidus necator.
  • the host of the transformed microorganism according to the present invention may be a wild strain originally having a PHA synthase gene, or a mutant strain obtained by artificially mutating such a wild strain. Alternatively, it may be a strain into which an exogenous PHA synthetase gene has been introduced by a genetic engineering method.
  • the method for introducing the foreign PHA synthase gene is not particularly limited, and the gene is directly inserted into the host chromosome. ⁇ 02020/174987 5 ⁇ (: 171?2020/003155
  • the method of directly inserting or substituting the gene into the megaplasmid possessed by the host or the method of placing the gene on the vector such as plasmid, phage, or phagemid and introducing it.
  • two or more of these methods may be used together.
  • a method of directly inserting or substituting the gene on the host chromosome or on the megaplasmid possessed by the host is preferable, and more preferably, on the chromosome of the host. This is a method of directly inserting or replacing the child.
  • the synthase gene is not particularly limited, but it comes from organisms belonging to the genus Ralstonia, genus Priavidas, genus Waltersia, genus Alcaligenes, genus Aeromonas, genus Cydmonas, genus Norcadia, and genus Chromobacterium! ! Examples include synthase genes and their variants. As the modified form, a base sequence encoding 1 to 18 synthetase in which one or more amino acid residues are deleted, added, inserted, or substituted can be used.
  • a gene having a nucleotide sequence encoding a polypeptide having 1 to 18 synthase activity is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more.
  • the types of 1 to 1 produced by the 2365 gene-enhanced strain of the present invention are not particularly limited as long as they can be produced by microorganisms!
  • Copolymers of two or more monomers selected from droxyalkanoic acid are preferred.
  • it is a homopolymer of 3-hydroxybutyric acid (abbreviation: 31 to 1 m) (31 to 1 m), a copolymer of 31 to 1 and 3-hydroxyvaleric acid (abbreviation: 31 to 1 V) (31 ⁇ 1 Snake one hundred-31 ⁇ 1) 31 - 1, M.
  • 3 HH 3 - hydroxy hexanoic acid
  • 3 HH 3 H Bc o-3 HH
  • PH BH 3 - hydroxy hexanoic acid
  • ⁇ 1 Snake and 1_ eighth copolymer (1_ eight thousand one hundred-31 ⁇ 1 snake) include, but are not limited to.
  • PH BH is preferable from the viewpoint of wide application range as a polymer.
  • the types 1 to 18 produced are the species of the Pl ⁇ A synthase gene possessed by the microorganism used or introduced separately, and the metabolic system involved in its synthesis. It can be appropriately selected depending on the type of gene, culture conditions and the like.
  • the 2365 gene of which expression is enhanced in the present invention is a polypeptide (II) which has the amino acid sequence of SEQ ID NO: 1. I 0 ⁇ ⁇ [ ⁇ 96
  • sequence homology is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more.
  • the function of the 8 2365 gene has not yet been reported.
  • the enhancement of gene expression in the present invention means a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is increased as compared with the strain in which the expression of the target gene is not enhanced. Point to.
  • the amount of increase is not particularly limited, it may be more than 1-fold, preferably 1.1-fold or more, more preferably 1.2-fold or more, as compared with the strain in which the expression of the target gene is not enhanced.
  • the increase is preferably 1.5 times or more, more preferably 2 times or more. ⁇ 02020/174987 7 ((171?2020/003155
  • the method for enhancing the expression of the 8 2 365 gene is not particularly limited, but the method of introducing the target gene into the host, the expression level of the target gene originally present in the genome lo You can choose to enhance, or both.
  • the method of introducing the target gene into the host is not particularly limited, but it may be a method of directly inserting or substituting the target gene into the dye of the host, or a method of directly inserting the target gene into the megaplasmid possessed by the host.
  • a method of substitution or a method of arranging and introducing the target gene on a vector such as plasmid, phage, or phagemid can be selected, and two or more of these methods may be used in combination.
  • a method of directly inserting or substituting the target gene on the host chromosome or on the megaplasmid possessed by the host is preferable, and more preferably on the host chromosome. This is a method of directly inserting or replacing the target gene.
  • the target gene is introduced so that it is located downstream of the “gene expression regulatory sequence” originally possessed by the host, or the target gene is a foreign “gene expression regulatory sequence”. It is preferable to introduce it in the form of being positioned downstream.
  • the “gene expression regulatory sequence” in the present invention refers to a nucleotide sequence that controls the transcription amount of the gene (for example, a promoter sequence), and/or a nucleotide sequence that regulates the translation amount of messenger 8 transcribed from the gene (for example, It is a mouth array including the Shine-Dalgarno array).
  • a promoter sequence for example, a promoter sequence
  • a nucleotide sequence that regulates the translation amount of messenger 8 transcribed from the gene for example, It is a mouth array including the Shine-Dalgarno array.
  • any base sequence existing in nature may be used, or an artificially constructed or modified base sequence may be used.
  • the method for enhancing the expression level of the target gene originally possessed by the host on the genome locus is not particularly limited, but a method for modifying a "gene expression regulatory sequence" located upstream of the target gene, For example, a method of introducing an exogenous "gene expression regulatory sequence" upstream of the gene, or a method of improving the stability of the transcribed messenger 8 by modifying the target gene and/or the nucleotide sequence around it. Can be mentioned.
  • the promoter sequence or Shine-Dalgarno sequence contained in the "gene expression regulatory sequence” includes, for example, the nucleotide sequences shown in any of SEQ ID NOs: 7 to 13 or a part of these nucleotide sequences. Examples include, but are not limited to, base sequences.
  • Substitution, deletion, insertion and/or addition of at least a part of genomic DNA can be performed by a method well known to those skilled in the art.
  • a method using the transposon and the mechanism of homologous recombination Ohman et al., J. BacterioL, 162:1068-1074 (1985)) or a site-specific mechanism that occurs by the mechanism of homologous recombination is used.
  • Method based on integration and loss by second-step homologous recombination (Noti et al.
  • RNA g RN A
  • the guide RNA g RN A
  • the guide RNA has a sequence that can bind to a part of the nucleotide sequence of the genome DN A to be modified, and plays a role in carrying C as 9 to the target.
  • the method of introducing the vector into the cells is not particularly limited, and examples thereof include a chlorinated sodium chloride method, an electro volatilization method, a polyethylene glycol method, and a spheroplast method.
  • P HA By culturing the A 2365 gene expression-enhancing strain of the present invention, P HA can be accumulated in the cells.
  • a conventional microorganism culture method can be used, and the culture may be performed in a medium in which an appropriate carbon source is present.
  • the medium composition, carbon source addition method, culture scale, aeration and stirring conditions, culture temperature, culture time, etc. are not particularly limited.
  • the carbon source is preferably added to the medium continuously or intermittently. ⁇ 02020/174987 9 ((171?2020/003155
  • any carbon source may be used as long as the strain capable of enhancing the gene expression of 2365 of the present invention can be assimilated.
  • sugars such as glucose, fructose and sucrose
  • palm oil and palm kernel oil including low melting point fractions such as palm olein, palm dull olein and palm kernel oil olein
  • Fats and oils such as corn oil, coconut oil, olive oil, soybean oil, rapeseed oil and jatropha oil, and their fractionated oils, or their refined by-products; lauric acid, oleic acid, stearic acid, palmitic acid, myristin Examples thereof include fatty acids such as acids and their derivatives, and glycerol.
  • gas and alcohols such as carbon dioxide, carbon dioxide, methane, methanol, and ethanol, these can also be used as carbon sources.
  • the microorganism is cultured using a medium containing the above carbon source, a nitrogen source which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources.
  • a nitrogen source which is a nutrient source other than the carbon source
  • inorganic salts and other organic nutrient sources.
  • the nitrogen source include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like.
  • the inorganic salts include potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like.
  • Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine, and proline, vitamins 1, vitamins 12, vitamins such as vitamin ⁇ , and the like.
  • the method of collection is not particularly limited.
  • cells are separated from the culture solution by a centrifuge or the like, dried, and then dried cells are washed with an organic solvent such as chloroform 1 to 1
  • the cell components are removed from the organic solvent solution containing !! by filtration, etc., and a poor solvent such as methanol or hexane is added to the filtrate to precipitate ! ⁇ 1, and the supernatant is collected by filtration or centrifugation.
  • the liquid can be removed and dried to recover !!. it can.
  • PHA PHA having a large particle size that can be produced according to the present invention, which is a preferred embodiment because it can be easily separated and recovered by such an aqueous system.
  • the overall genetic manipulation can be performed as described in, for example, M ol e c u l a r C l o n i n g (Co l d S p r i n g H a r b o r L a b o r a t o r y P r e s s (1 989)).
  • enzymes used for genetic engineering, cloning hosts, etc. can be purchased from market suppliers and used according to the instructions. The enzyme is not particularly limited as long as it can be used for gene manipulation.
  • the plasmid pCUP2-trc-A2365 for A2365 gene expression was prepared. The production was performed as follows.
  • Capriavidas necatol H was detected by PCR using synthetic oligo DNA.
  • a DN A fragment (SEQ ID NO: 14) having an A 2365 gene sequence was obtained by using the genome D NA of 16 strains as a hook type. This DNA fragment was digested with the restriction enzymes Mu n and S pel, and the resulting DNA fragment was cloned into the plasmid pCU P 2 described in WO 2007/0497 16 in Mu n and S pe. It was connected with the one cut in to obtain Plasmid vector _ p CU P 2-A2365.
  • a DN A fragment (SEQ ID NO: 15) having a promoter sequence derived from Escherichia coli was obtained by PCR using a synthetic oligo DN A. This DNA fragment was digested with restriction enzymes Ec RI and Mun, and the obtained DNA fragment was ligated with the plasmid vector pCU P2-A 2365 cleaved with Mun. The A2365 gene sequence was promoted from the obtained plasmid vector. ⁇ 02020/174987 11 ⁇ (: 171? 2020 /003155
  • the plasmid vector linked in the direction downstream of the Yuichi sequence was selected and designated as 8365-expressing plasmid vector ⁇ 11 2-I “ ⁇ -8 23 65.
  • a plasmid vector for expression of the 8 2365 gene ⁇ 11 2_ I “ ⁇ -8 8365 was introduced into the ⁇ 1 ⁇
  • the resulting transformant can be prepared according to the method described in US Pat. No. 7,384,766.
  • the introduction of plasmid vector into cells was carried out by electrical introduction as follows.
  • the gene transfer device used was a Gene Pulser from 3 companies, and the Kyubet was a 98 0.20 from Io V ⁇ 6 as well.
  • An electric pulse was applied under the conditions of a capacitance of 25, a voltage of 1.51 ⁇ , and a resistance value of 8000.
  • the pulse was injected into the cuvette.
  • the bacterial solution of 1 ⁇ ! r ⁇ & ⁇ t 3 " ⁇ medium manufactured by 0 I ⁇ Company
  • composition of the seed culture medium is as follows: ⁇ / ⁇ / / V% 1 ⁇ /1631: _6 Father 1: "3:1:, 1 3 ⁇ 4//%% ⁇ ⁇ ⁇ — I-"So 1:0 3 ⁇ 4 //% ⁇ 6 3 3 1: — ⁇ Father
  • composition of the pre-culture medium is 1.1 ⁇ «/% 3 2 1 ⁇ 1 ? ⁇ .1 21 ⁇ 1 2 0, 0 .19 ⁇ 02020/174987 12 ((171?2020/003155
  • Palm olein oil was added as a carbon source at a concentration of 109/1_ all at once.
  • the ratio of accumulated amount was measured as follows. The cells were recovered from the culture solution by centrifugation, washed with ethanol, freeze-dried, and the dried cells were collected and weighed. Chloroform of 100 cc was added to the obtained dried cells 19 and the mixture was stirred at room temperature for 24 hours to extract 1 to 1 in the cells. After filtering the bacterial cell residue, it was concentrated in an evaporator until the total volume reached 3001 liters, 9001 I hexane was gradually added, and the mixture was left for 1 hour with slow stirring. After filtering out the deposited ! ⁇ 18,
  • the particle size of 1 to 18 particles was measured as follows. After culturing, the culture solution is treated at 65 ° ⁇ for 60 minutes to inactivate the microbial cells, then diluted 150-fold with 3.3 /% sodium dodecyl sulfate aqueous solution, and ultrasonically disrupted! An extract was obtained. For ultrasonic crushing, use 3 IV! Dingsha's ultrasonic disperser 111 ⁇ 1? 600, and ⁇ 02020/174987 13 13 (: 171?2020/003155
  • the treatment was performed 4 times for 0 seconds.
  • the obtained 1 ⁇ 1 extract is laser diffraction scattering type particle size distribution measuring device 1//1 3300 ⁇ 11 1) was analyzed and the volume average diameter (IV! V) of ! ⁇ ! eight particles was measured.
  • the measurement was carried out with standard settings (particle permeability: transmission, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index:... 333).
  • the 1 to 18 production culture was performed as follows. First, 005 shares of glycero
  • Lustock (50 ⁇ ) to seed medium Were inoculated into the seeds and cultured for 24 hours to perform seed culture.
  • the seed culture medium ... . 31_ jar fermenter with 81_ pre-culture medium (Maruhishi Bioengine 1_-300 type)! . ⁇ / ⁇ % inoculated.
  • the operating conditions were a culture temperature of 33 °0, a stirring speed of 500 ", an aeration rate of 1.8 L/min, and 1 to 1 was controlled between 6.7 and 6.8 for 28 hours, and preculture For 1 to 1 control, a 14% aqueous solution of ammonium hydroxide was used.
  • the preculture liquid was added to a 5 !_ jar fermenter (manufactured by Maruhishi Bio Engineering Co., Ltd.) containing 1 to 18 production medium of 2.5 !_ Was vaccinated with 5.0 /%.
  • the operating conditions culture temperature 33 ° 0, stirring speed 420 ", aeration rate 2.
  • 1 1_ / Rei_1 ⁇ 1 ⁇ 1 to 1 was controlled between 6.7 to 6.8.
  • 1-1 Control the mouth Lumpur using 25% aqueous ammonium hydroxide solution.
  • the carbon source as the intermittently added. carbon source was used palm olein oil.
  • culture the ratio of 1 to 1 accumulated amount for dry cells weight
  • the ratio of the accumulated amount of 1 to 18 and the particle size of 1 to 1 were measured as described above, and the results are shown in Table 1.
  • Table 1 shows the measurement results of the 1 to 1 accumulation ratio and the 1 to 1 particle size.
  • the particle size of 1 to 18 produced by the 2365 gene expression-enhanced strain was 8 to the 2365 gene expression-unenhanced strain. ⁇ 02020/174987 Compared with the particle size of 1 to 1 produced by 14 strains (:171?2020/003155 strain), an increase was recognized.

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Abstract

A transgenic microorganism having a PHA synthetase gene and enhanced in the expression of A2365 gene. A method for producing PHA, the method comprising the step of culturing the transgenic microorganism in the presence of a carbon source.

Description

\¥02020/174987 1 ?€1/^2020/003155 \¥02020/174987 1 ?€1/^2020/003155
明 細 書 Specification
発明の名称 ： Title of invention:
形質転換微生物、 及びポリヒドロキシアルカン酸の製造方法 Transformed microorganism and method for producing polyhydroxyalkanoic acid
技術分野 Technical field
[0001 ] 本発明は、 ポリヒドロキシアルカン酸を生産可能な形質転換微生物、 及び 、 当該形質転換微生物を用いたポリヒドロキシアルカン酸の製造方法に関す る。 The present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.
背景技術 Background technology
[0002] 環境問題、 食糧問題、 健康及び安全に対する意識の高まり、 天然又は自然 志向の高まりなどを背景に、 微生物を利用した物質製造 （発酵生産、 バイオ 変換など） の意義及び重要性が益々高まっており、 タンパク質医薬品や遺伝 子治療用の核酸などの製造にも、 微生物による物質生産が応用されている。 例えば、 酵母やバクテリアなどの微生物を利用したエタノール、 酢酸、 医療 用タンパク質の生産などが活発に産業応用されている。 [0002] Against the backdrop of environmental problems, food problems, increased awareness of health and safety, and increased awareness of nature or nature, the significance and importance of substance production using microorganisms (fermentation production, bioconversion, etc.) is increasing. The production of substances by microorganisms is also applied to the production of protein drugs and nucleic acids for gene therapy. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria are actively being applied industrially.
[0003] その一例として、 生分解性プラスチックとしての産業利用が期待されてい るポリヒドロキシアルカン酸 （以下、 !·!八ともいう） の微生物による生産 が挙げられる （非特許文献 1 を参照） 。 !~1八は、 多くの微生物種の細胞に エネルギー蓄積物質として産生、 蓄積される熱可塑性ポリエステルであり、 生分解性を有している。 現在、 環境への意識の高まりから非石油由来のブラ スチックが注目されるなか、 特に、 微生物が菌体内に産生、 蓄積する !·!八 は、 自然界の炭素循環プロセスに取り込まれることから生態系への悪影響が 小さいと予想されており、 その実用化が切望されている。 微生物を利用した |_|八生産では、 例えば、 カプリアビダス属細菌に炭素源として糖、 植物油 脂や脂肪酸を与え、 細胞内に 1~1八を蓄積させることで 1~1八を生産するこ とが知られている （非特許文献 2及び 3を参照） 。 [0003] As an example thereof, there is a microbial production of polyhydroxyalkanoic acid (hereinafter also referred to as !8!), which is expected to be industrially used as a biodegradable plastic (see Non-Patent Document 1). ! ~ 18 is a thermoplastic polyester that is produced and accumulated in cells of many microbial species as an energy storage substance and has biodegradability. At present, non-petroleum-derived plastics are attracting attention due to increasing environmental awareness.In particular, microorganisms are produced and accumulated in the fungus body!...8 because they are incorporated into the carbon cycle process in the natural world. It is expected that the adverse effect on the product will be small, and its practical application is highly desired. Using microorganisms | _ | was eight production, for example, sugars, vegetable oil fat or fatty acid given as a carbon source in Cupriavidus bacterium, a child produces 1-1 eight by to accumulate 1 to 1 (viii) in the intracellular Are known (see Non-Patent Documents 2 and 3).
[0004] しかしながら、 微生物を利用した物質生産においては、 目的生産物の分離 回収工程が煩雑となり、 生産コストが高くなることが問題になるケースがあ \¥02020/174987 2 卩（：171?2020/003155 [0004] However, in the production of substances using microorganisms, there are cases in which the separation and recovery process of the target product becomes complicated and the production cost becomes high. \¥02020/174987 2 (:171?2020/003155
る。 従って、 目的生産物の分離回収効率を向上させることは、 生産コストの 低減のための大きな課題である。 It Therefore, improving the separation and recovery efficiency of the target product is a major issue to reduce the production cost.
[0005] 非特許文献 4には、 カプリアビダス属細菌においてフェイシンタンパク質 をコードする遺伝子 II 3 1 を破壊することで、 非破壊の場合より大粒子 径の 1~1 を蓄積したことが報告されており、 これを目的生産物の分離回収 効率を改善する方法として採用することが考えられる。 しかし、
Figure imgf000003_0001
[0005] Non-Patent Document 4 reports that disruption of the gene II 31 encoding the fasin protein in a bacterium belonging to the genus Capriavidus accumulated a larger particle size of 1 to 1 than in the case of non-disruption. Therefore, it is possible to adopt this as a method to improve the separation and recovery efficiency of the target product. But,
Figure imgf000003_0001
1破壊株は P HA蓄積量が著しく減少することが示されており、 工業生産に 適したものではなかった。 The 1-disrupted strain was shown to significantly reduce the amount of PHA accumulated, and was not suitable for industrial production.
先行技術文献 Prior art documents
非特許文献 Non-patent literature
[0006] 非特許文献 1 : 八门〇16 「 3〇门 八」 . ，
Figure imgf000003_0002
a . , 1 门 1: . 」 . 巳 1[0006] Non-Patent Document 1: 8 门 〇 16 "30 门 8" .,
Figure imgf000003_0002
a ., 1 门 1:. 」. 巳 1
〇 I . 1\/18〇 1^〇 〇 1. ， 1 2， 1 02- 1 05 ( 1 990) ○ I .1\/18 ○ 1^ ○ ○ 1., 1 2, 1 02- 1 05 (1 990)
非特許文献 2 : 38 I 〇 3. ， ㊀ 8 し ， 」 6 I 〇 5〇 I . 6 I 〇 6 门 . ， 1 20 (3) ， 246-25 1 (201 5) Non-Patent Document 2: 38 I ○ 3., ㊀ 8 and ”6 I ○ 5 ○ I .6 I ○ 6 doors., 1 20 (3), 246-25 1 (201 5)
非特許文献 3 : 1 门 3〇111 11リ门 0 ， ㊀ a 1\/16 813· 巳门 . ， 27， 38-45 (201 5) Non-Patent Document 3:1 1 3 0 111 11 Re 0, ㊀ a 1\/16 813·Mimi.
非特許文献
Figure imgf000003_0003
IV!. ， ø I a 1\/1 1 〇 1"〇 1〇 1 〇 1 〇 9 V , 1 5 1 (9 I 3) ， 825-833 (2005) 発明の概要 Non-patent literature
Figure imgf000003_0003
IV!., ø I a 1\/1 1 ○ 1" ○ 1 ○ 1 ○ 1 ○ 9 V, 1 5 1 (9 I 3), 825-833 (2005) Summary of the invention
発明が解決しようとする課題 Problems to be Solved by the Invention
[0007] 1~1 は微生物細胞内において粒子状に蓄積される。 微生物細胞内に蓄積 された 1~1八を生分解性プラスチックとして利用するためには、 細胞を破砕 して !·! 粒子を取り出し、 他の細胞成分から分離し、 回収する。 分離回収 の手法は、 大きくは有機溶媒系による方法と水系による方法に分けられるが 、 有機溶媒の使用は高環境負荷、 高コストとなるため、 工業的には水系によ る方法が好ましい。 水系による方法では、 例えば、 !·!八粒子を含む細胞破 砕液から、 遠心分離機や分離膜等によって !~1 粒子を分離することができ \¥02020/174987 3 卩（：171?2020/003155 [0007] 1 to 1 is accumulated in the particulate in the microbial cells. In order to use 1 to 18 accumulated in microbial cells as a biodegradable plastic, the cells are crushed to extract the particles, and the particles are separated from other cell components and collected. The method of separation and recovery can be roughly classified into an organic solvent-based method and an aqueous method, but the use of an organic solvent has a high environmental load and is costly, and therefore the aqueous method is industrially preferable. In the water-based method, for example, ! ~ 1 particles can be separated from the cell lysate containing !8 particles using a centrifuge or a separation membrane. \¥02020/174987 3 ((171?2020/003155
る。 このような場合、 分離回収の効率は 1~1 粒子の大きさに依存すること になる。 即ち、 微生物細胞内に蓄積された 1~1 粒子が大きいほど、 遠心分 離機や分離膜等を用いた分離回収を容易に実施でき、 生産コストの低減につ ながる。 It In such cases, the efficiency of separation and recovery depends on the size of 1 to 1 particles. That is, the larger the 1 to 1 particle accumulated in the microbial cell, the easier the separation and recovery using a centrifugal separator or a separation membrane, which leads to a reduction in production cost.
[0008] 本発明は、 上記現状に鑑み、 大粒子径の
Figure imgf000004_0001
を蓄積可能な形質転換微生 物、 及び、 当該形質転換微生物を用いた 1~1 の製造方法を提供することを 課題とする。 [0008] In view of the above situation, the present invention has
Figure imgf000004_0001
It is an object of the present invention to provide a transformed microorganism capable of accumulating a bacterium, and a method for producing 1 to 1 using the transformed microorganism.
課題を解決するための手段 Means for solving the problem
[0009] 本発明者らは鋭意検討した結果、 カプリアビダス属細菌において機能未知 とされている八2 3 6 5遺伝子 （例えば配列番号 1 に記載のアミノ酸配列を コードする遺伝子） の発現を強化することで、 工業的に望ましい !·!八蓄積 量を維持しながら、 微生物細胞内に蓄積される 1~1 の粒子径を拡大できる ことを見出し、 本発明に至った。 [0009] As a result of diligent studies, the present inventors have found that enhancement of the expression of the 8 2 365 gene (for example, the gene encoding the amino acid sequence set forth in SEQ ID NO: 1) whose function is unknown in the bacteria of the genus Capriavidus Thus, the inventors have found that the particle size of 1 to 1 accumulated in microbial cells can be increased while maintaining the industrially desirable accumulation amount, and the present invention has been completed.
[0010] すなわち本発明は、 ポリヒドロキシアルカン酸合成酵素遺伝子を有し、 八 That is, the present invention has a polyhydroxyalkanoic acid synthase gene,
2 3 6 5遺伝子の発現が強化された、 形質転換微生物に関する。 前記形質転 換微生物は、 カプリアビダス属に属することが好ましく、 カプリアビダス - ネカ卜ールの形質転換微生物であることがより好ましい。 The present invention relates to a transformed microorganism having enhanced expression of 2365 gene. The transformed microorganisms preferably belong to the genus Capriavidas, and more preferably, the transformed microorganisms of Capriavidus-neca laurel.
また本発明は、 前記形質転換微生物を、 炭素源の存在下で培養する工程を 含む、 ポリヒドロキシアルカン酸の製造方法にも関する。 炭素源は、 油脂あ るいは脂肪酸を含有してもよく、 また、 糖を含有してもよく、 二酸化炭素を 含有してもよい。 ポリヒドロキシアルカン酸は、 2種以上のヒドロキシアル カン酸の共重合体であることが好ましく、 3 -ヒドロキシヘキサン酸をモノ マーユニッ トとして含有する共重合体であることがより好ましく、 3 -ヒド ロキシ酪酸と 3 -ヒドロキシヘキサン酸との共重合体であることがさらに好 ましい。 The present invention also relates to a method for producing polyhydroxyalkanoic acid, which comprises a step of culturing the transformed microorganism in the presence of a carbon source. The carbon source may contain fats or oils or fatty acids, may contain sugar, and may contain carbon dioxide. The polyhydroxyalkanoic acid is preferably a copolymer of two or more kinds of hydroxyalkane acids, more preferably a copolymer containing 3-hydroxyhexanoic acid as a monomer unit, and 3-hydroxy More preferably, it is a copolymer of butyric acid and 3-hydroxyhexanoic acid.
発明の効果 Effect of the invention
[001 1 ] 本発明によれば、 大粒子径の !·! を蓄積可能な形質転換微生物、 及び、 当該形質転換微生物を用いた ! ! の製造方法を提供することができる。 本 発明によると、 微生物細胞内に大粒子径の P H A粒子が蓄積されるため、 細 胞破砕後に細胞成分からの P H Aの分離回収が容易となり、 生産コストの低 減を実現することができる。 [001 1] According to the present invention, it is possible to provide a transformed microorganism capable of accumulating a large number of particles!, and a method for producing !! using the transformed microorganism. Book According to the invention, since PHA particles having a large particle size are accumulated in the microbial cells, it becomes easy to separate and recover PHA from the cell components after cell disruption, and it is possible to reduce the production cost.
発明を実施するための形態 MODE FOR CARRYING OUT THE INVENTION
[0012] 以下、 本発明の実施形態を詳細に説明する。 [0012] Hereinafter, embodiments of the present invention will be described in detail.
本発明に係る形質転換微生物は、 P H A合成酵素遺伝子を有し、 A236 5遺伝子の発現が強化された、 形質転換微生物である。 The transformed microorganism according to the present invention is a transformed microorganism having a PHA synthase gene and enhanced expression of the A2365 gene.
[0013] (微生物) [0013] (Microorganism)
本発明に係る形質転換微生物は、 P H A合成酵素遺伝子を有し、 かつ、 A 2365遺伝子の発現が強化されるように形質転換された微生物である (以 下、 A 2365遺伝子発現強化株ともいう) 。 本発明に係る形質転換微生物 の宿主は、 P H A合成酵素遺伝子を有する微生物であれば特に限定されない 。 当該細菌としては、 例えば、 ラルストニア (R a l s t o n i a) 属、 力 プリアビダス (C u p r i a v i d u s) 属、 ワウテルシア (W a u t e r s i a) 属、 アエロモナス (Ae r omo n a s) 属、 エシエリキア (E s c h e r i c h i a) 属、 アルカリゲネス (A l c a l i g e n e s) 属、 シュードモナス (P s e u d omo n a s) 属等に属する細菌類が好ましい 例として挙げられる。 安全性及び P H A生産性の観点から、 より好ましくは ラルストニア属、 カプリアビダス属、 アエロモナス属、 ワウテルシア属に属 する細菌であり、 さらに好ましくはカプリアビダス属又はアエロモナス属に 属する細菌であり、 さらにより好ましくはカプリアビダス属に属する微生物 であり、 特に好ましくはカプリアビダス ネカトール (C u p r i a V i d u s n e c a t o r) である。 The transformed microorganism according to the present invention has a PHA synthase gene, and is a microorganism transformed so that the expression of the A 2365 gene is enhanced (hereinafter, also referred to as A 2365 gene expression-enhanced strain). .. The host of the transformed microorganism of the present invention is not particularly limited as long as it is a microorganism having a PHA synthase gene. Examples of the bacterium include Ralstonia genus, Cupriavidus genus, W autersia genus, Aeromonas (Ae r omo nas) genus, Escherichia genus, Alcaligenes (A lcaligenes). Preferable examples include bacteria belonging to the genus Pseud omo nas and the like. From the viewpoints of safety and PHA productivity, bacteria more preferably belonging to the genus Ralstonia, the genus Capriavidus, the genus Aeromonas, and the genus Woutercia are more preferred, and the bacteria belonging to the genus Capriavidus or the genus Aeromonas are still more preferred, and still more preferably capriavidus. It is a microorganism belonging to the genus, and particularly preferably Capriavidus necator.
[0014] 本発明に係る形質転換微生物の宿主は、 P H A合成酵素遺伝子を本来的に 有する野生株であってもよいし、 そのような野生株を人工的に突然変異処理 して得られる変異株や、 あるいは、 遺伝子工学的手法により外来の P H A合 成酵素遺伝子が導入された菌株であってもよい。 外来の P H A合成酵素遺伝 子を導入する方法は特に限定されず、 宿主の染色体上に遺伝子を直接揷入ま \¥02020/174987 5 卩（：171?2020/003155 [0014] The host of the transformed microorganism according to the present invention may be a wild strain originally having a PHA synthase gene, or a mutant strain obtained by artificially mutating such a wild strain. Alternatively, it may be a strain into which an exogenous PHA synthetase gene has been introduced by a genetic engineering method. The method for introducing the foreign PHA synthase gene is not particularly limited, and the gene is directly inserted into the host chromosome. \¥02020/174987 5 卩 (: 171?2020/003155
たは置換する方法、 宿主が保有するメガプラスミ ド上に遺伝子を直接挿入ま たは置換する方法、 あるいはプラスミ ド、 ファージ、 ファージミ ドなどのべ クター上に遺伝子を配置して導入する方法などが選択でき、 これらの方法の うち 2つ以上を併用しても良い。 導入遺伝子の安定性を考慮すると、 好まし くは、 宿主の染色体上または宿主が保有するメガプラスミ ド上に遺伝子を直 接揷入または置換する方法であり、 より好ましくは、 宿主の染色体上に遺伝 子を直接揷入または置換する方法である。 Or the method of substitution, the method of directly inserting or substituting the gene into the megaplasmid possessed by the host, or the method of placing the gene on the vector such as plasmid, phage, or phagemid and introducing it. Yes, two or more of these methods may be used together. Considering the stability of the transgene, a method of directly inserting or substituting the gene on the host chromosome or on the megaplasmid possessed by the host is preferable, and more preferably, on the chromosome of the host. This is a method of directly inserting or replacing the child.
[0015] （ 1~1八合成酵素遺伝子） [0015] (1 to 1 synthase gene)
1~1八合成酵素遺伝子としては特に限定されないが、 ラルストニア属、 力 プリアビダス属、 ワウテルシア属、 アルカリゲネス属、 アエロモナス属、 シ ュードモナス属、 ノルカディア属、 クロモバクテリウム属に類する生物に由 来する !·! 合成酵素遺伝子や、 それらの改変体などが挙げられる。 前記改 変体としては、 1以上のアミノ酸残基が欠失、 付加、 挿入、 又は置換された 1~1八合成酵素をコードする塩基配列などを用いることができる。 例えば、 配列番号 2〜 6のいずれかに記載のアミノ酸配列で示されるポリべプチドを コードする塩基配列を有する遺伝子、 及び、 該アミノ酸配列に対して 8 5 % 以上の配列相同性を有するアミノ酸配列で示され、 かつ 1~1八合成酵素活性 を有するポリべプチドをコードする塩基配列を有する遺伝子などが挙げられ る。 上記配列相同性としては好ましくは 9 0 %以上、 より好ましくは 9 5 % 以上、 さらに好ましくは 9 7 %以上、 特に好ましくは 9 9 %以上である。 1 to 18 The synthase gene is not particularly limited, but it comes from organisms belonging to the genus Ralstonia, genus Priavidas, genus Waltersia, genus Alcaligenes, genus Aeromonas, genus Cydmonas, genus Norcadia, and genus Chromobacterium! ! Examples include synthase genes and their variants. As the modified form, a base sequence encoding 1 to 18 synthetase in which one or more amino acid residues are deleted, added, inserted, or substituted can be used. For example, a gene having a nucleotide sequence encoding the polypeptide represented by the amino acid sequence of any of SEQ ID NOs: 2 to 6, and an amino acid sequence having 85% or more sequence homology to the amino acid sequence. And a gene having a nucleotide sequence encoding a polypeptide having 1 to 18 synthase activity. The sequence homology is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more.
[0016] （P l·\ A） [0016] (P l·\ A)
本発明の 2 3 6 5遺伝子発現強化株が生産する 1~1 の種類としては、 微生物が生産し得る !·!八である限り特に限定されないが、 炭素数 4〜 1 6 の 3—ヒドロキシアルカン酸から選択される 1種のモノマーの単独重合体、 炭素数 4〜 1 6の 3—ヒドロキシアルカン酸から選択される 1種のモノマー とその他のヒドロキシアルカン酸 （例えば、 炭素数 4〜 1 6の 2 -ヒドロキ シアルカン酸、 4 -ヒドロキシアルカン酸、 5 -ヒドロキシアルカン酸、 6 —ヒドロキシアルカン酸など） の共重合体、 及び、 炭素数 4〜 1 6の 3—ヒ \¥02020/174987 6 卩（：171?2020/003155 The types of 1 to 1 produced by the 2365 gene-enhanced strain of the present invention are not particularly limited as long as they can be produced by microorganisms! A homopolymer of one monomer selected from acids, one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (for example, having 4 to 16 carbon atoms). A copolymer of 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc., and 3-hydroxyamine having 4 to 16 carbon atoms \¥02020/174987 6 卩 (: 171?2020/003155
ドロキシアルカン酸から選択される 2種以上のモノマーの共重合体が好まし い。 例えば、 3 -ヒドロキシ酪酸 （略称： 31~1巳） のホモポリマーである （31~1巳） 、 31~1巳と 3 -ヒドロキシ吉草酸 （略称： 31~1 V） の共重合体 （31~1巳一〇〇-31~1 ） 、 31~1巳と 3 -ヒドロキシヘキサン酸 （略称： 3 H H） の共重合体 （3 H B-c o-3 HH） （略称： PH BH） 、 31~1巳 と 4 -ヒドロキシ酪酸 （略称： 41~1巳） の共重合体 （3 H B-c o-4 H 巳） 、 乳酸 （略称： 1_八） を構成成分として含む !·!八、 例えば 31~1巳と 1_ 八の共重合体 （1_八一〇〇-31~1巳） などが挙げられるが、 これらに限定 されない。 この中でも、 ポリマーとしての応用範囲が広いという観点から、 PH BHが好ましい。 なお、 生産される 1~1八の種類は、 目的に応じて、 使 用する微生物の保有するあるいは別途導入された P l·\A合成酵素遺伝子の種 類や、 その合成に関与する代謝系の遺伝子の種類、 培養条件などによって適 宜選択しうる。 Copolymers of two or more monomers selected from droxyalkanoic acid are preferred. For example, it is a homopolymer of 3-hydroxybutyric acid (abbreviation: 31 to 1 m) (31 to 1 m), a copolymer of 31 to 1 and 3-hydroxyvaleric acid (abbreviation: 31 to 1 V) (31 ~ 1 Snake one hundred-31 ~ 1) 31 - 1, M. and 3 - hydroxy hexanoic acid (abbreviation: 3 HH) of the copolymer (3 H Bc o-3 HH) (abbreviation: PH BH) 31 - A copolymer of 1-min and 4-hydroxybutyric acid (abbreviation: 41 to 1-min) (3 H Bco-4 H-min) and lactic acid (abbreviation: 1_8) as constituents! such as ~ 1 Snake and 1_ eighth copolymer (1_ eight thousand one hundred-31 ~ 1 snake) include, but are not limited to. Among these, PH BH is preferable from the viewpoint of wide application range as a polymer. Depending on the purpose, the types 1 to 18 produced are the species of the Pl·\A synthase gene possessed by the microorganism used or introduced separately, and the metabolic system involved in its synthesis. It can be appropriately selected depending on the type of gene, culture conditions and the like.
[0017] （八2365遺伝子） [0017] (8 2365 genes)
本発明で発現が強化される 2365遺伝子は、 配列番号 1 に記載のアミ ノ酸配列で示されるポリペプチド （II门 丨
Figure imgf000007_0001
I 0 〇〇 [<96The 2365 gene of which expression is enhanced in the present invention is a polypeptide (II) which has the amino acid sequence of SEQ ID NO: 1.
Figure imgf000007_0001
I 0 〇 〇 [<96
1） 、 及び、 該アミノ酸配列に対して 85%以上の配列相同性を有するアミ ノ酸配列で示されるポリべプチドをコードする塩基配列を有する遺伝子であ る。 上記配列相同性としては好ましくは 90 %以上、 より好ましくは 95 % 以上、 さらに好ましくは 97 %以上、 特に好ましくは 99 %以上である。 な お、 八 2365遺伝子が有する機能は未だ報告されていない。 1) and a gene having a nucleotide sequence encoding a polypeptide represented by an amino acid sequence having 85% or more sequence homology with the amino acid sequence. The sequence homology is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more. The function of the 8 2365 gene has not yet been reported.
[0018] （遺伝子発現強化） [0018] (Enhanced gene expression)
本発明における遺伝子発現の強化とは、 対象遺伝子の発現が強化されてい ない菌株と比較して、 対象遺伝子の転写量または対象遺伝子のコードするポ リぺプチドの発現量が増加している状態を指す。 その増加量は特に限定され ないが、 対象遺伝子の発現が強化されていない菌株と比較して 1倍超であれ ばよく、 好ましくは 1. 1倍以上、 より好ましくは 1. 2倍以上、 さらに好 ましくは 1. 5倍以上、 さらにより好ましくは 2倍以上の増加である。 \¥02020/174987 7 卩（：171?2020/003155 The enhancement of gene expression in the present invention means a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is increased as compared with the strain in which the expression of the target gene is not enhanced. Point to. Although the amount of increase is not particularly limited, it may be more than 1-fold, preferably 1.1-fold or more, more preferably 1.2-fold or more, as compared with the strain in which the expression of the target gene is not enhanced. The increase is preferably 1.5 times or more, more preferably 2 times or more. \¥02020/174987 7 ((171?2020/003155
[0019] 本発明において、 八 2 3 6 5遺伝子の発現を強化する方法は特に限定され ないが、 対象遺伝子を宿主に導入する方法、 宿主がゲノムロ 上に元来有 する対象遺伝子の発現量を増強する方法、 またはその両方を選択することが できる。 [0019] In the present invention, the method for enhancing the expression of the 8 2 365 gene is not particularly limited, but the method of introducing the target gene into the host, the expression level of the target gene originally present in the genome lo You can choose to enhance, or both.
[0020] 対象遺伝子を宿主に導入する方法としては特に限定されないが、 宿主の染 色体上に対象遺伝子を直接挿入または置換する方法、 宿主が保有するメガプ ラスミ ド上に対象遺伝子を直接揷入または置換する方法、 あるいはプラスミ ド、 ファージ、 ファージミ ドなどのべクター上に対象遺伝子を配置して導入 する方法などが選択でき、 これらの方法のうち 2つ以上を併用しても良い。 [0020] The method of introducing the target gene into the host is not particularly limited, but it may be a method of directly inserting or substituting the target gene into the dye of the host, or a method of directly inserting the target gene into the megaplasmid possessed by the host. Alternatively, a method of substitution or a method of arranging and introducing the target gene on a vector such as plasmid, phage, or phagemid can be selected, and two or more of these methods may be used in combination.
[0021 ] 導入遺伝子の安定性を考慮すると、 好ましくは、 宿主の染色体上または宿 主が保有するメガプラスミ ド上に対象遺伝子を直接挿入または置換する方法 であり、 より好ましくは、 宿主の染色体上に対象遺伝子を直接揷入または置 換する方法である。 導入する遺伝子を確実に発現させるために、 対象遺伝子 が、 宿主が元来有する 「遺伝子発現調節配列」 の下流に位置するように導入 するか、 または、 対象遺伝子が、 外来の 「遺伝子発現調節配列」 の下流に位 置する形で導入することが好ましい。 本発明における 「遺伝子発現調節配列 」 とは、 その遺伝子の転写量を制御する塩基配列 （例えばプロモーター配列 ） 、 及び/または、 その遺伝子から転写されたメッセンジャー 八の翻訳 量を調節する塩基配列 （例えばシャイン ·ダルガノ配列） を含む口 配列 である。 「遺伝子発現調節配列」 としては、 自然界に存在する任意の塩基配 列を利用することもできるし、 人工的に構築または改変された塩基配列を利 用しても良い。 [0021] Considering the stability of the transgene, a method of directly inserting or substituting the target gene on the host chromosome or on the megaplasmid possessed by the host is preferable, and more preferably on the host chromosome. This is a method of directly inserting or replacing the target gene. In order to ensure the expression of the introduced gene, the target gene is introduced so that it is located downstream of the “gene expression regulatory sequence” originally possessed by the host, or the target gene is a foreign “gene expression regulatory sequence”. It is preferable to introduce it in the form of being positioned downstream. The “gene expression regulatory sequence” in the present invention refers to a nucleotide sequence that controls the transcription amount of the gene (for example, a promoter sequence), and/or a nucleotide sequence that regulates the translation amount of messenger 8 transcribed from the gene (for example, It is a mouth array including the Shine-Dalgarno array). As the "gene expression regulatory sequence", any base sequence existing in nature may be used, or an artificially constructed or modified base sequence may be used.
[0022] また、 宿主がゲノムロ 八上に元来有する対象遺伝子の発現量を増強する 方法としては特に限定されないが、 対象遺伝子の上流に位置する 「遺伝子発 現調節配列」 を改変する方法、 対象遺伝子の上流に外来の 「遺伝子発現調節 配列」 を導入する方法、 あるいは、 対象遺伝子及び/またはその周辺の塩基 配列を改変することにより、 転写されたメッセンジャー 八の安定性を向 上させる方法などが挙げられる。 [0023] 「遺伝子発現調節配列」 に含まれるプロモーター配列やシャイン ·ダルガ ノ配列としては、 例えば、 配列番号 7〜 1 3のいずれかに示される塩基配列 、 または、 これら塩基配列の一部を含む塩基配列などが挙げられるが、 特に 限定されない。 [0022] Further, the method for enhancing the expression level of the target gene originally possessed by the host on the genome locus is not particularly limited, but a method for modifying a "gene expression regulatory sequence" located upstream of the target gene, For example, a method of introducing an exogenous "gene expression regulatory sequence" upstream of the gene, or a method of improving the stability of the transcribed messenger 8 by modifying the target gene and/or the nucleotide sequence around it. Can be mentioned. [0023] The promoter sequence or Shine-Dalgarno sequence contained in the "gene expression regulatory sequence" includes, for example, the nucleotide sequences shown in any of SEQ ID NOs: 7 to 13 or a part of these nucleotide sequences. Examples include, but are not limited to, base sequences.
[0024] ゲノム DN Aの少なくとも一部の置換、 欠失、 揷入及び/又は付加は、 当 業者に周知の方法により行うことができる。 代表的な方法としてはトランス ポゾンと相同組換えの機構を利用した方法 (Ohman et al., J. BacterioL, 162:1068-1074 (1985)) や、 相同組換えの機構によって起こる部位特異的な 組み込みと第二段階の相同組換えによる脱落を原理とした方法 (Noti et al. [0024] Substitution, deletion, insertion and/or addition of at least a part of genomic DNA can be performed by a method well known to those skilled in the art. As a typical method, a method using the transposon and the mechanism of homologous recombination (Ohman et al., J. BacterioL, 162:1068-1074 (1985)) or a site-specific mechanism that occurs by the mechanism of homologous recombination is used. Method based on integration and loss by second-step homologous recombination (Noti et al.
, Methods EnzymoL, 154:197-217 (1987)) などがある。 また、 Bacillus su bti Us由来の sacB遺伝子を共存させて、 第二段階の相同組換えによって遺伝 子が脱落した微生物株をスクロース耐性株として容易に単離する方法 (Schwe izer, Mol. Microbiol. , 6: 1195-1204 (1992)、 Lenz et a 1. , J. Bacter i o 1., Methods EnzymoL, 154:197-217 (1987)). In addition, a method in which the sacB gene from Bacillus su bti Us is allowed to coexist and a microbial strain from which the gene has been eliminated by the second-step homologous recombination is easily isolated as a sucrose resistant strain (Schweizer, Mol. 6: 1195-1204 (1992), Lenz et a 1., J. Bacter io 1.
, 176:4385-4393 (1994)) も利用することができる。 さらに別の方法として 、 標的 D N Aを改変するための C R I S P R/C a s 9システムによるゲノ ム編集技術 (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) も利 用することができる。 C R 丨 S P R/C a s 9システムでは、 ガイ ド R N A (g RN A) は改変すべきゲノム DN Aの塩基配列の一部に結合しうる配列 を有しており、 C a s 9を標的に運ぶ役割をもつ。 , 176:4385-4393 (1994)) can also be used. As another method, a genomic editing technology using the CRISPR/C as 9 system for modifying target DNA (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) is also used. can do. In the CR SPR/C as 9 system, the guide RNA (g RN A) has a sequence that can bind to a part of the nucleotide sequence of the genome DN A to be modified, and plays a role in carrying C as 9 to the target. With.
[0025] 細胞へのベクターの導入方法としても特に限定されないが、 例えば、 塩化力 ルシウム法、 エレクトロボレーシヨン法、 ポリエチレングリコール法、 スフ エロプラスト法等が挙げられる。 [0025] The method of introducing the vector into the cells is not particularly limited, and examples thereof include a chlorinated sodium chloride method, an electro volatilization method, a polyethylene glycol method, and a spheroplast method.
[0026] 本発明の A 2365遺伝子発現強化株を培養することで、 菌体内に P HA を蓄積させることができる。 本発明の A 2365遺伝子発現強化株を培養す る方法としては、 常法の微生物培養法に従うことができ、 適切な炭素源が存 在する培地中で培養を行なえばよい。 培地組成、 炭素源の添加方法、 培養ス ケール、 通気攪拌条件や、 培養温度、 培養時間などは特に限定されない。 炭 素源は、 連続的に、 または間欠的に培地に添加することが好ましい。 \¥02020/174987 9 卩（：171?2020/003155 By culturing the A 2365 gene expression-enhancing strain of the present invention, P HA can be accumulated in the cells. As a method for culturing the A 2365 gene expression-enhanced strain of the present invention, a conventional microorganism culture method can be used, and the culture may be performed in a medium in which an appropriate carbon source is present. The medium composition, carbon source addition method, culture scale, aeration and stirring conditions, culture temperature, culture time, etc. are not particularly limited. The carbon source is preferably added to the medium continuously or intermittently. \¥02020/174987 9 ((171?2020/003155
[0027] 培養時の炭素源としては、 本発明の 2 3 6 5遺伝子発現強化株が資化可 能であればどのような炭素源でも使用可能である。 特に限定されないが、 例 えば、 グルコース、 フルクトース、 シュークロースなどの糖類；パーム油や パーム核油 （これらを分別した低融点分画であるパームオレイン、 パームダ ブルオレイン、 パーム核油オレインなども含む） 、 コーン油、 やし油、 オリ —ブ油、 大豆油、 菜種油、 ヤトロファ油などの油脂やその分画油類、 あるい はその精製副産物； ラウリン酸、 オレイン酸、 ステアリン酸、 パルミチン酸 、 ミリンスチン酸などの脂肪酸やそれらの誘導体、 あるいはグリセロール等 が挙げられる。 また、 本発明の 2 3 6 5遺伝子発現強化株が二酸化炭素、 —酸化炭素、 メタン、 メタノール、 エタノールなどのガスやアルコール類を 利用可能である場合、 これらを炭素源として使用することもできる。 [0027] As the carbon source during culture, any carbon source may be used as long as the strain capable of enhancing the gene expression of 2365 of the present invention can be assimilated. For example, sugars such as glucose, fructose and sucrose; palm oil and palm kernel oil (including low melting point fractions such as palm olein, palm dull olein and palm kernel oil olein) are not particularly limited. Fats and oils such as corn oil, coconut oil, olive oil, soybean oil, rapeseed oil and jatropha oil, and their fractionated oils, or their refined by-products; lauric acid, oleic acid, stearic acid, palmitic acid, myristin Examples thereof include fatty acids such as acids and their derivatives, and glycerol. In addition, when the 2365 gene-enhanced strain of the present invention is able to use gas and alcohols such as carbon dioxide, carbon dioxide, methane, methanol, and ethanol, these can also be used as carbon sources.
[0028] 本発明における !·! の製造では、 上記炭素源、 炭素源以外の栄養源であ る窒素源、 無機塩類、 その他の有機栄養源を含む培地を用いて、 前記微生物 を培養することが好ましい。 下記に限定されないが、 窒素源としては、 例え ば、 アンモニア；塩化アンモニウム、 硫酸アンモニウム、 リン酸アンモニウ ム等のアンモニウム塩；ペプトン、 肉エキス、 酵母エキス等が挙げられる。 無機塩類としては、 例えば、 リン酸 2水素カリウム、 リン酸水素 2ナトリウ ム、 リン酸マグネシウム、 硫酸マグネシウム、 塩化ナトリウム等が挙げられ る。 その他の有機栄養源としては、 例えば、 グリシン、 アラニン、 セリン、 スレオニン、 プロリン等のアミノ酸、 ビタミン巳 1、 ビタミン巳 1 2、 ビタ ミン〇等のビタミン等が挙げられる。 [0028] In the production of !... In the present invention, the microorganism is cultured using a medium containing the above carbon source, a nitrogen source which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Is preferred. Examples of the nitrogen source include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like. Examples of the inorganic salts include potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like. Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine, and proline, vitamins 1, vitamins 12, vitamins such as vitamin 〇, and the like.
[0029] 培養を適切な時間行なって菌体内に 1~1八を蓄積させた後、 周知の方法を 用いて菌体から 1~1八を回収する。 回収方法については特に限定されないが 、 例えば、 培養終了後、 培養液から遠心分離機等で菌体を分離し、 乾燥させ た後、 乾燥菌体から、 クロロホルム等の有機溶剤を用いて 1~1 を抽出し、 この ! ! を含んだ有機溶剤溶液から濾過等によって細胞成分を除去し、 そ の濾液にメタノールやヘキサン等の貧溶媒を加えて !~1 を沈殿させ、 濾過 や遠心分離によって上澄み液を除去し、 乾燥させて ! ! を回収することが できる。 また、 界面活性剤やアルカリ、 酵素などを用いて P H A以外の細胞 成分を水に溶解させた後、 濾過や遠心分離によって P H A粒子を水相から分 離し乾燥させて回収することもできる。 本発明により製造され得る大粒径の P HA 、 このような水系による分離回収が容易に実施できるため好ましい 実施例 [0029] After performing the appropriate time to accumulate 1-1 (viii) in the bacterial cells and culture, recovering 1-1-eight cells using well known methods. The method of collection is not particularly limited.For example, after the culture is completed, cells are separated from the culture solution by a centrifuge or the like, dried, and then dried cells are washed with an organic solvent such as chloroform 1 to 1 The cell components are removed from the organic solvent solution containing !! by filtration, etc., and a poor solvent such as methanol or hexane is added to the filtrate to precipitate ! ~ 1, and the supernatant is collected by filtration or centrifugation. The liquid can be removed and dried to recover !!. it can. It is also possible to dissolve the cellular components other than PHA in water using a surfactant, an alkali, an enzyme, etc., and then separate the PHA particles from the aqueous phase by filtration and centrifugation to dry and recover them. PHA having a large particle size that can be produced according to the present invention, which is a preferred embodiment because it can be easily separated and recovered by such an aqueous system.
[0030] 以下、 実施例により本発明をさらに具体的に説明する。 ただし、 本発明は 、 これら実施例に限定されるものではない。 なお全体的な遺伝子操作は、 例 えば M o l e c u l a r C l o n i n g （Co l d S p r i n g H a r b o r L a b o r a t o r y P r e s s （1 989） ） に記載され ているように行うことができる。 また、 遺伝子操作に使用する酵素、 クロー ニング宿主等は、 市場の供給者から購入し、 その説明に従い使用することが できる。 なお、 酵素としては、 遺伝子操作に使用できるものであれば特に限 定されない。 [0030] Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be performed as described in, for example, M ol e c u l a r C l o n i n g (Co l d S p r i n g H a r b o r L a b o r a t o r y P r e s s (1 989)). In addition, enzymes used for genetic engineering, cloning hosts, etc. can be purchased from market suppliers and used according to the instructions. The enzyme is not particularly limited as long as it can be used for gene manipulation.
[0031] （製造例） A 2365遺伝子発現強化株の作製 (Production Example) Preparation of A 2365 gene expression enhanced strain
まず、 A 2365遺伝子発現用プラスミ ド pCU P 2- t r c - A 236 5の作製を行った。 作製は以下のように行った。 First, the plasmid pCUP2-trc-A2365 for A2365 gene expression was prepared. The production was performed as follows.
[0032] 合成オリゴ DNAを用いた PCRにより、 カプリアビダス ·ネカトール H [0032] Capriavidas necatol H was detected by PCR using synthetic oligo DNA.
1 6株のゲノム D N Aを錶型として、 A 2365遺伝子配列を有する DN A 断片 （配列番号 1 4） を得た。 この DN A断片を制限酵素 Mu n 丨 および S p e lで消化し、 得られた DNA断片を、 国際公開 2007 /0497 1 6 号に記載のプラスミ ドべクター pCU P 2を Mu n 丨 および S p e 丨で切断 したものと連結して、 プラスミ ドべクタ _ p CU P 2-A2365を得た。 A DN A fragment (SEQ ID NO: 14) having an A 2365 gene sequence was obtained by using the genome D NA of 16 strains as a hook type. This DNA fragment was digested with the restriction enzymes Mu n and S pel, and the resulting DNA fragment was cloned into the plasmid pCU P 2 described in WO 2007/0497 16 in Mu n and S pe. It was connected with the one cut in to obtain Plasmid vector _ p CU P 2-A2365.
[0033] さらに、 合成オリゴ DN Aを用いた PC Rにより、 大腸菌に由来するプロ モーター配列を有する DN A断片 （配列番号 1 5） を得た。 この DNA断片 を制限酵素 E c〇 R I および M u n 丨で消化し、 得られた D N A断片を、 プ ラスミ ドべクター pCU P 2— A 2365を Mu n 丨で切断したものと連結 した。 得られたプラスミ ドベクターから、 A 2365遺伝子配列がプロモー \¥02020/174987 11 卩（：171? 2020 /003155 [0033] Furthermore, a DN A fragment (SEQ ID NO: 15) having a promoter sequence derived from Escherichia coli was obtained by PCR using a synthetic oligo DN A. This DNA fragment was digested with restriction enzymes Ec RI and Mun, and the obtained DNA fragment was ligated with the plasmid vector pCU P2-A 2365 cleaved with Mun. The A2365 gene sequence was promoted from the obtained plasmid vector. \¥02020/174987 11 卩 (: 171? 2020 /003155
夕一配列の下流側に位置する向きで連結されたプラスミ ドべクターを選別し 、 八2365遺伝子発現用プラスミ ドベクター 〇11 2- I 「〇-八23 65とした。 The plasmid vector linked in the direction downstream of the Yuichi sequence was selected and designated as 8365-expressing plasmid vector 〇 11 2-I “〇-8 23 65.
[0034] 次に、 八2365遺伝子発現用プラスミ ドベクター 〇11 2_ I 「〇— 八2365を＜1\| [＜_005株に導入して、 八 2365遺伝子発現強化株を 得た。
Figure imgf000012_0001
株は、 カプリアビダス ネカトール!· I 1 6株の染色体上 にアエロモナス ·キヤビエ由来の 1~1 合成酵素遺伝子 （配列番号 4に記載 のアミノ酸配列を有する Pl·\A合成酵素をコードする遺伝子） が導入された 形質転換体であり、 米国特許第 7384766号明細書に記載の方法に準じ て作成することができる。 [0034] Next, a plasmid vector for expression of the 8 2365 gene 〇 11 2_ I “○-8 8365 was introduced into the <1\| [<_005 strain to obtain the 8 2365 gene expression-enhanced strain.
Figure imgf000012_0001
Strain, Cupriavidus Nekatoru! - (gene encoding Pl-\ A synthetase having the amino acid sequence set forth in SEQ ID NO: 4) 1-1 synthase gene derived from Aeromonas Kiyabie to I 1 on chromosome six strains introduced The resulting transformant can be prepared according to the method described in US Pat. No. 7,384,766.
[0035] プラスミ ドべクターの細胞への導入は以下のように電気導入によって行っ た。 遺伝子導入装置は巳 丨 〇 「 3 社製のジーンパルサーを用い、 キユべッ 卜は同じく巳 I 〇 V ^ 6社製の 98 0. 2〇 を用いた。 キユベッ トに、 コンビテント細胞 40〇 丨 と発現べクター 2〇 丨 を注入してパルス装置 にセッ トし、 静電容量 25 、 電圧 1. 51＜ 、 抵抗値 8000の条件で 電気パルスをかけた。 パルス後、 キユべッ ト内の菌液を 1\!リ r \ & 〇 t 3 「〇 培地 （0 I 〇〇社製） で 30°〇、 3時間振とう培養し、 選択プレ —卜 （1\!リ
Figure imgf000012_0002
〇〇社製） 、 カナマイシン 1 00 9/1_） で、 30°〇にて 2日間培養して、 生育してきた八 2365遺 伝子発現強化株を取得した。 [0035] The introduction of plasmid vector into cells was carried out by electrical introduction as follows. The gene transfer device used was a Gene Pulser from 3 companies, and the Kyubet was a 98 0.20 from Io V ^ 6 as well. An electric pulse was applied under the conditions of a capacitance of 25, a voltage of 1.51<, and a resistance value of 8000. The pulse was injected into the cuvette. The bacterial solution of 1 \! r \ & 〇 t 3 "○ medium (manufactured by 0 I 〇 Company) was shake-cultured at 30° 〇 for 3 hours, and the pre-selected (1\!
Figure imgf000012_0002
KK), kanamycin 100 9 /1_), and cultured for 2 days at 30° 〇 to obtain the 8365 gene expression-enhanced strain that had grown.
[0036] （比較例）
Figure imgf000012_0003
(Comparative Example)
Figure imgf000012_0003
下記の条件で
Figure imgf000012_0004
[＜_005株を用いた培養検討を行なった。 Under the following conditions
Figure imgf000012_0004
[<-005 strains were used for culture studies.
[0037] （培地） [0037] (Media)
種母培地の組成は飞 \/\/ / V % 1\/1631： _6父 1： 「 3〇 1：、 1 ¾// ％ 巳 〇 〇— I - 「 ソ 1： 0 门 6、 0 2 ¾// ％ 丫 6 3 3 1： —㊀ 父 「 The composition of the seed culture medium is as follows: \/\/ / V% 1\/1631: _6 Father 1: "3:1:, 1 ¾//%% 〇 〇 〇 — I-"So 1:0 ¾ //% 丫 6 3 3 1: — ㊀ Father
9 / ％ 321~1 〇4 - 1 21~12〇 、 〇. 1 5 / V% ＜9 /% 3 2 1-1 〇 4 -. 1 21-1 2 〇, 〇 1 5 / V% <
1~12?〇4、 （ 1~16. 8） とした。 It was set to 1 ~ 1 2 ~ 0 4 , (1 ~ 16.8).
[0038] 前培養培地の組成は 1. 1 \«/ ％ 321~1 ?〇 . 1 21~12〇、 〇. 1 9 \¥02020/174987 12 卩（：171?2020/003155
Figure imgf000013_0001
[0038] The composition of the pre-culture medium is 1.1 \ «/% 3 2 1 ~ 1 ?○ .1 21 ~ 1 2 0, 0 .19 \¥02020/174987 12 ((171?2020/003155
Figure imgf000013_0001
% IV! 93〇4 71~12〇、 2. 5 / ％ パームオレインオイル、 0. 5 ▽ /V% 微量金属塩溶液 （〇. 1 1\1塩酸に ·！ . 6\/\// ％ 6〇 1 3 - 6% IV! 93 〇 4 71 ~ 1 2 〇, 2.5 /% Palm olein oil, 0.5 ▽ /V% Trace metal salt solution (〇.1 1\1 Hydrochloric acid! % 6_Rei 1 3 - 6
1~12〇、 1 / ％ 〇 3〇 1 2 - 21~12〇、 〇. 02 / ％ 0〇 0 I 2 1-1 2 〇, 1 /% 〇 3_Rei 1 2 -. 21-1 2 〇, 〇 02 /% 0_Rei 0 I 2
61~12〇、 〇. 01 6 / ％ 〇リ 3〇4 51~12〇、 〇. 01 2 / ％61 ~ 1 2 〇, 〇 .01 6 /% 〇 Re 3 〇 4 51 ~ 1 2 〇, 〇. 01 2 /%
1\1 I 〇 丨 2 61~12〇を溶かしたもの） とした。 炭素源としてパームオレイン オイルを 1 〇 9/1_の濃度で一括添加した。 1\1 I 〇 丨2 61 ~ 1 2 〇 melted). Palm olein oil was added as a carbon source at a concentration of 109/1_ all at once.
[0039] 1~1八生産培地の組成は〇. 385 / V% 321~1 ?〇4 . 1 21~12〇、
Figure imgf000013_0002
[0039] 1 to eight composition of the production medium 〇. 385 / V% 3 2 1 ~ 1? 〇 4.1 21-1 2 〇,
Figure imgf000013_0002
. 1 / ％ IV! 93〇4 71~12〇、 〇. 5 V / V % 微量金属塩溶液 （0 . 1 1\1塩酸に ·！ . 6 / ％ 6〇 1 3 - 61~12〇、 1 / ％ 030 I 2 21~12〇、 〇. 02 / ％ 〇〇〇 1 2 - 61~12〇、 〇. 01 6 / ％.!. 1 /% IV 93_Rei 4 71-1 2 〇, 〇 5 V / V% trace metal salt solution (0 1 1 \ - 1 HCl 6 /% 6_Rei 1 3 -.!. 61-1 2 〇, 1 /% 030 I 2 21 ~ 1 2 〇, 〇 02 /% thousand 1 2 -.. 61-1 2 〇, 〇 01 6 /%
〇リ 3〇 4 - 51~12〇、 〇. 01 2 / ％ 1\1 I 〇 I 2 61~12〇を溶かした もの） とした。 〇ri 3 〇 4-51 ~ 1 2 〇, 〇 .01 2 /% 1\1 I 〇 I 2 61 〜 1 2 〇).
[0040] （ ? !!八蓄積量割合の測定方法） [0040] (?!! Eight accumulated amount measurement method)
!! 蓄積量の割合は次のように測定した。 遠心分離によって培養液から 菌体を回収、 エタノールで洗浄、 凍結乾燥し、 乾燥菌体を取得し、 重量を測 定した。 得られた乾燥菌体 1 9に 1 〇〇 丨のクロロホルムを加え、 室温で 一昼夜攪拌して、 菌体内の 1~1 を抽出した。 菌体残渣をろ別後、 エバポレ —夕一で総容量が 3001 丨 になるまで濃縮後、 9001 Iのヘキサンを徐々に 加え、 ゆっくり攪拌しながら、 1時間放置した。 析出した !~1八をろ別後、The ratio of accumulated amount was measured as follows. The cells were recovered from the culture solution by centrifugation, washed with ethanol, freeze-dried, and the dried cells were collected and weighed. Chloroform of 100 cc was added to the obtained dried cells 19 and the mixture was stirred at room temperature for 24 hours to extract 1 to 1 in the cells. After filtering the bacterial cell residue, it was concentrated in an evaporator until the total volume reached 3001 liters, 9001 I hexane was gradually added, and the mixture was left for 1 hour with slow stirring. After filtering out the deposited ! ~ 18,
50°◦で 3時間真空乾燥した。 乾燥 !·! の重量を測定し、 乾燥菌体量に対 して 1~1八蓄積量が占める割合を算出した。 Vacuum dried at 50° for 3 hours. The dry matter was weighed, and the ratio of the accumulated amount of 1 to 18 to the dry cell amount was calculated.
[0041] （ 1~1八粒子径の測定方法） [0041] (1 to 8 particle size measuring method)
1~1八粒子径は次のように測定した。 培養後の培養液を 65 °〇で 60分間 処理し、 菌体細胞不活化を行った後、 3. 3 / ％ ドデシル硫酸ナトリ ウム水溶液により 1 50倍に希釈し、 超音波破砕により !·! 抽出液を得た 。 超音波破砕には 3 IV!丁社製超音波分散機111~1? 600を用い、 最大出力で 4 \¥02020/174987 13 卩（：171?2020/003155 The particle size of 1 to 18 particles was measured as follows. After culturing, the culture solution is treated at 65 ° 〇 for 60 minutes to inactivate the microbial cells, then diluted 150-fold with 3.3 /% sodium dodecyl sulfate aqueous solution, and ultrasonically disrupted! An extract was obtained. For ultrasonic crushing, use 3 IV! Dingsha's ultrasonic disperser 111 ~ 1? 600, and \¥02020/174987 13 13 (: 171?2020/003155
0秒、 4回の処理を行った。 得られた 1~1 抽出液をレーザー回折 ·散乱式 粒子径分布測定装置
Figure imgf000014_0001
1\/1丁 3300巳乂 1 1） により 解析し、 !·!八粒子の体積平均径 （IV! V） を測定した。 測定は標準的な設定 （粒子透過性：透過、 粒子屈折率： 1. 81、 粒子形状：非球形、 溶媒屈折 率： ·！ . 333） で行った。 The treatment was performed 4 times for 0 seconds. The obtained 1 ~ 1 extract is laser diffraction scattering type particle size distribution measuring device
Figure imgf000014_0001
1//1 3300 跳乂11 1) was analyzed and the volume average diameter (IV! V) of !·! eight particles was measured. The measurement was carried out with standard settings (particle permeability: transmission, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index:... 333).
[0042] （ !·!八生産培養） [0042] (!·! eight production culture)
1~1八生産培養は次のように行った。 まず、
Figure imgf000014_0002
005株のグリセロThe 1 to 18 production culture was performed as follows. First,
Figure imgf000014_0002
005 shares of glycero
—ルストック （ 50 丨） を種母培地
Figure imgf000014_0003
に接種して 24時間培養 し種母培養を行なった。 次に種母培養液を、 ·！ . 81_の前培養培地を入れた 31_ジャーファーメンター （丸菱バイオエンジ製
Figure imgf000014_0004
1_-300型） に ·！ . 〇▽/▽%接種した。 運転条件は、 培養温度 33 °0、 攪拌速度 500 「 、 通気量 1. 8 L/m i nとし、 1~1は 6. 7〜 6. 8の間でコントロール しながら 28時間培養し、 前培養を行なった。 1~1コントロールには 1 4 % 水酸化アンモニウム水溶液を使用した。 — Lustock (50 丨) to seed medium
Figure imgf000014_0003
Were inoculated into the seeds and cultured for 24 hours to perform seed culture. Next, the seed culture medium... . 31_ jar fermenter with 81_ pre-culture medium (Maruhishi Bioengine
Figure imgf000014_0004
1_-300 type)! .○▽/▽% inoculated. The operating conditions were a culture temperature of 33 °0, a stirring speed of 500 ", an aeration rate of 1.8 L/min, and 1 to 1 was controlled between 6.7 and 6.8 for 28 hours, and preculture For 1 to 1 control, a 14% aqueous solution of ammonium hydroxide was used.
[0043] 次に、 前培養液を、 2. 5 !_の 1~1八生産培地を入れた 5 !_ジャーファー メンター （丸菱バイオエンジ製
Figure imgf000014_0005
に 5. 〇 / ％接種し た。 運転条件は、 培養温度 33°0、 攪拌速度 420 「 、 通気量 2. 1 1_ /〇1 丨 1^とし、 1~1は 6. 7〜 6. 8の間でコントロールした。 1~1コント 口ールには 25%水酸化アンモニウム水溶液を使用した。 炭素源は断続的に 添加した。 炭素源としてはパームオレインオイルを使用した。 培養は、 乾燥 菌体量に対する 1~1 蓄積量の割合が 90%程度に達するまで行った。 1~1 八蓄積量の割合および 1~1 粒子径は前述のように測定した。 結果を表 1 に 示す。 [0043] Next, the preculture liquid was added to a 5 !_ jar fermenter (manufactured by Maruhishi Bio Engineering Co., Ltd.) containing 1 to 18 production medium of 2.5 !_
Figure imgf000014_0005
Was vaccinated with 5.0 /%. The operating conditions, culture temperature 33 ° 0, stirring speed 420 ", aeration rate 2. 1 1_ / Rei_1丨1 ^ 1 to 1 was controlled between 6.7 to 6.8. 1-1 Control the mouth Lumpur using 25% aqueous ammonium hydroxide solution. the carbon source as the intermittently added. carbon source was used palm olein oil. culture, the ratio of 1 to 1 accumulated amount for dry cells weight The ratio of the accumulated amount of 1 to 18 and the particle size of 1 to 1 were measured as described above, and the results are shown in Table 1.
[0044] （実施例） 八 2365遺伝子発現強化株による !!八生産 (Example) Eight 2365 gene expression-enhanced strains!! Eight production
比較例と同様の条件で 2365遺伝子発現強化株を用いた培養検討を行 なった。 1~1 蓄積量の割合および 1~1 粒子径の測定結果を表 1 に示す。 Under the same conditions as in Comparative Example, a culture study was carried out using a 2365 gene expression-enhanced strain. Table 1 shows the measurement results of the 1 to 1 accumulation ratio and the 1 to 1 particle size.
[0045] 培養検討の結果、 2365遺伝子発現強化株によって生産された 1~1八 の粒子径は、 八 2365遺伝子の発現を強化していない株 — 005 \¥02020/174987 14 卩（：171?2020/003155 株） によって生産された 1~1 の粒子径と比較して、 増大が認められた。 [0045] As a result of the culture examination, the particle size of 1 to 18 produced by the 2365 gene expression-enhanced strain was 8 to the 2365 gene expression-unenhanced strain. \02020/174987 Compared with the particle size of 1 to 1 produced by 14 strains (:171?2020/003155 strain), an increase was recognized.
[0046] なお、 比較例および実施例の培養検討によって生産された 1~1 は 1~1巳
Figure imgf000015_0001
分析にて確認した。 [0046] In addition, 1 to 1 produced by the culture studies of Comparative Examples and Examples is 1 to 1
Figure imgf000015_0001
Confirmed by analysis.
[0047] [表 1 ] [0047] [Table 1]
Figure imgf000015_0002
Figure imgf000015_0002

Claims

\¥02020/174987 15 卩（：171?2020/003155 請求の範囲 \¥02020/174987 15 ((171?2020/003155 Claims
[請求項 1 ] ポリヒドロキシアルカン酸合成酵素遺伝子を有し、 八2 3 6 5遺伝 子の発現が強化された、 形質転換微生物。 [Claim 1] A transformed microorganism having a polyhydroxyalkanoic acid synthase gene and enhanced expression of 8 2 3 6 5 genes.
[請求項 2] カプリアビダス属に属する、 請求項 1 に記載の形質転換微生物。 [Claim 2] The transformed microorganism according to claim 1, which belongs to the genus Capriavidus.
[請求項 3] カプリアビダス ·ネカトールの形質転換微生物である、 請求項 2に 記載の形質転換微生物。 [Claim 3] The transformed microorganism according to claim 2, which is a transformed microorganism of Capriavidus necatol.
[請求項 4] 請求項 1〜 3のいずれか 1項に記載の形質転換微生物を、 炭素源の 存在下で培養する工程を含む、 ポリヒドロキシアルカン酸の製造方法 [Claim 4] A method for producing polyhydroxyalkanoic acid, which comprises a step of culturing the transformed microorganism according to any one of claims 1 to 3 in the presence of a carbon source.
[請求項 5] 炭素源が、 油脂あるいは脂肪酸を含有する、 請求項 4に記載のポリ ヒドロキシアルカン酸の製造方法。 [Claim 5] The method for producing a polyhydroxyalkanoic acid according to claim 4, wherein the carbon source contains fats and oils or fatty acids.
[請求項 6] 炭素源が、 糖を含有する、 請求項 4に記載のポリヒドロキシアルカ ン酸の製造方法。 [Claim 6] The method for producing a polyhydroxyalkanoic acid according to claim 4, wherein the carbon source contains a sugar.
[請求項 7] 炭素源が、 二酸化炭素を含有する、 請求項 4に記載のポリヒドロキ シアルカン酸の製造方法。 [Claim 7] The method for producing a polyhydroxyalkanoic acid according to claim 4, wherein the carbon source contains carbon dioxide.
[請求項 8] ポリヒドロキシアルカン酸が、 2種以上のヒドロキシアルカン酸の 共重合体である、 請求項 4〜 7のいずれか 1項に記載のポリヒドロキ シアルカン酸の製造方法。 [Claim 8] The method for producing a polyhydroxyalkanoic acid according to any one of claims 4 to 7, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid.
[請求項 9] 前記ポリヒドロキシアルカン酸が、 3 -ヒドロキシヘキサン酸をモ ノマーユニッ トとして含有する共重合体である、 請求項 8に記載のポ リヒドロキシアルカン酸の製造方法。 9. The method for producing polyhydroxyalkanoic acid according to claim 8, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.
[請求項 10] 前記ポリヒドロキシアルカン酸が、 3 -ヒドロキシ酪酸と 3 -ヒド ロキシヘキサン酸との共重合体である、 請求項 9に記載のポリヒドロ キシアルカン酸の製造方法。 10. The method for producing polyhydroxyalkanoic acid according to claim 9, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.
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