WO2023085374A1 - ポリヒドロキシアルカン酸の製造方法 - Google Patents

ポリヒドロキシアルカン酸の製造方法 Download PDF

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WO2023085374A1
WO2023085374A1 PCT/JP2022/041970 JP2022041970W WO2023085374A1 WO 2023085374 A1 WO2023085374 A1 WO 2023085374A1 JP 2022041970 W JP2022041970 W JP 2022041970W WO 2023085374 A1 WO2023085374 A1 WO 2023085374A1
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pha
enzyme
production method
microorganism
cells
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French (fr)
Japanese (ja)
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佳弘 毛利
尚志 有川
俊輔 佐藤
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Kaneka Corp
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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a method for producing polyhydroxyalkanoic acid.
  • microorganisms a substance production by microorganisms is also applied to the production of protein pharmaceuticals and nucleic acids for gene therapy.
  • substance production by microorganisms is also applied to the production of protein pharmaceuticals and nucleic acids for gene therapy.
  • the production of ethanol, acetic acid, and proteins for medical use using microorganisms such as yeast and bacteria is actively applied industrially.
  • PHA polyhydroxyalkanoic acid
  • PHA polyhydroxyalkanoic acid
  • Non-Patent Document 1 PHA is a biodegradable thermoplastic polyester that is produced and accumulated in the cells of many microbial species as an energy storage substance.
  • non-petroleum-derived plastics are attracting attention due to growing environmental awareness.
  • PHA which is produced and accumulated in the cells of microorganisms, is taken into the carbon cycle process of the natural world and has an adverse effect on the ecosystem. It is expected to be small, and its practical application is earnestly desired.
  • Non-Patent Document 2 In PHA production using microorganisms, for example, it is known that PHA is produced by supplying sugar, vegetable oil or fatty acid as a carbon source to bacteria of the genus Capriavidus and accumulating PHA in cells (Non-Patent Document 2). and 3).
  • PHAs produced by microorganisms are usually accumulated in the form of granules within the cells of the microorganisms. Therefore, in order to use PHAs as plastics, it is necessary to separate and recover the PHAs from the cells of the microorganisms. be. Further, in order to use PHA as a plastic, it is desirable to increase the purity of PHA and reduce the content of contaminants such as bacterial cell constituents as much as possible.
  • Patent Literatures 1 and 2 disclose a method of combining a treatment for crushing PHA-containing microbial cells and a surfactant treatment, a method of adding an alkali and performing a heat treatment followed by a crushing treatment, and the like.
  • Patent Document 3 discloses a method for obtaining PHA by treating an aqueous suspension of microbial cells with sodium hypochlorite, an enzyme, or the like to solubilize biological components other than PHA.
  • methods for recovering PHA from the resulting aqueous suspension include, for example, separation operations such as centrifugation and filtration, and spraying.
  • separation operations such as centrifugation and filtration, and spraying.
  • a drying operation using a dryer, a drum dryer, or the like can be mentioned.
  • the PHA concentration of the aqueous suspension is high in order to dry efficiently.
  • the higher the PHA concentration the lower the fluidity of the aqueous suspension and the more difficult the drying operation.
  • Patent Document 4 discloses a method of achieving good dispersibility by adding polyvinyl alcohol as a dispersant to a PHA aqueous dispersion.
  • the addition of dispersants may affect the processability of the recovered PHA, so addition of large amounts is undesirable.
  • Optimizing the shape of the particles contained in the aqueous suspension is conceivable as a means of improving the fluidity of the aqueous suspension while maintaining the concentration of the aqueous suspension at a high level.
  • the particle shape that provides the highest fluidity of the aqueous suspension is spherical.
  • the fluidity of the aqueous suspension tends to decrease as the surface area per unit volume of the particles increases, such as elliptical, plate-like, rod-like, and rod-like.
  • the shape of PHA particles depends on the shape of the microbial cells that accumulate the particles. In many cases, the shape of microbial cells with sufficient PHA accumulation is spherical. For example, when PHA is accumulated to about 90% in the KNK-005 strain, the cells become spherical, and the PHA particles obtained by crushing the cells are also spherical (see Reference Example 1 described later).
  • the shape of PHA-accumulating cells may not be spherical.
  • the cell division inhibitory enzyme minCD is overexpressed or some cell wall degrading enzyme genes are disrupted as in Patent Documents 5 and 6, PHA-accumulating cells become rod-shaped. PHA particles that accumulate in rod-shaped cells tend to be rod-shaped. Therefore, an aqueous PHA suspension obtained from rod-shaped PHA-accumulating cells tends to have low fluidity.
  • the object of the present invention is to provide a method for producing PHA that enables the collection of PHA particles that are more nearly spherical from rod-shaped PHA-accumulating cells.
  • the present inventors have discovered that when rod-shaped microbial cells that accumulate PHA are treated with a specific enzyme, the PHA particles become spherical, leading to the present invention.
  • the present invention comprises a step of culturing a polyhydroxyalkanoic acid-producing microorganism to obtain microbial cells that have accumulated polyhydroxyalkanoic acid, and a step of treating the microbial cells with an enzyme, wherein the microbial cells have a cell diameter of
  • the present invention relates to a method for producing a polyhydroxyalkanoic acid having a number average aspect ratio of 2.0 or more, wherein the enzyme includes a cell wall degrading enzyme.
  • aqueous suspension containing the PHA particles can exhibit relatively high fluidity even if the PHA concentration is high because the PHA particles are nearly spherical. Therefore, the efficiency in the subsequent drying process and the like can be enhanced.
  • An embodiment of the present invention relates to a method for producing PHA, comprising the steps of culturing a PHA-producing microorganism to obtain microbial cells of a specific shape that accumulate PHA, and treating the microbial cells with an enzyme.
  • PHA The type of PHA is not particularly limited as long as it is a PHA that can be produced by microorganisms. It may be a homopolymer composed of one hydroxyalkanoic acid, or composed of two or more hydroxyalkanoic acids. It may be a copolymer to be used.
  • a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, and other of hydroxyalkanoic acid e.g., 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid having 4 to 16 carbon atoms, etc.
  • hydroxyalkanoic acid e.g., 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid having 4 to 16 carbon atoms, etc.
  • 4 to 16 carbon atoms and copolymers of two or more monomers selected from 3-hydroxyalkanoic acids e.g., 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid having 4 to 16 carbon atoms, etc.
  • homopolymers or copolymers containing 3-hydroxybutyric acid as a monomer unit are preferred.
  • polymers include P(3HB), which is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), and P(3HB-co), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV).
  • the type of PHA to be produced is appropriately selected according to the purpose, depending on the type of PHA synthase gene possessed by the microorganism to be used or separately introduced, the type of metabolic system gene involved in its synthesis, culture conditions, etc. I can.
  • the PHA-producing microorganism may be any microorganism capable of producing PHA.
  • the microorganism may be a microorganism having a PHA synthase gene.
  • the microorganism may be a wild strain that originally has a PHA synthase gene, a mutant strain obtained by artificially mutating such a wild strain, or a gene It may be a strain into which an exogenous PHA synthase gene has been introduced by engineering techniques.
  • the PHA-producing microorganism has a non-spherical cell shape, preferably a rod-like shape, when it accumulates PHA.
  • the PHA-producing microorganism may be a wild strain whose cell shape is non-spherical when PHA is accumulated, or may be a wild strain whose cell shape is non-spherical when PHA is accumulated by artificial mutation treatment or genetic engineering techniques. It may be a strain mutated to be non-spherical.
  • Examples include gene recombination and gene recombination that reduces the expression of the A1386 gene and/or the 2405 gene, which is a gene presumed to be a peptidoglycan hydrolase. These genetically modified strains can change the cell shape to non-spherical. However, gene recombination in which the cell shape changes to non-spherical is not limited to these examples.
  • the effect of the invention can be obtained by interrupting the culture at the stage when the cell shape is non-spherical and performing the enzymatic treatment according to the present disclosure.
  • the PHA-producing microorganism or the host of the microorganism is not particularly limited, but is preferably a bacillus, more preferably a Gram-negative bacillus.
  • the bacilli include bacteria belonging to the family Burkholderiaceae, such as the genus Ralstonia, the genus Cupriavidus, the genus Wautersia, and the genus Burkholderia, and Pseudomonas.
  • Preferable examples include bacteria belonging to the genus, bacteria belonging to the genus Halomonas, and bacteria belonging to the genus Escherichia.
  • bacteria belonging to the genus Ralstonia, Capriavidus, or Escherichia are more preferred, bacteria belonging to the genus Capriavidus or Escherichia are more preferred, and Capriavidus necator is particularly preferred. (Cupriavidus necator) or Escherichia coli.
  • PHA synthase gene The PHA synthase gene retained by the PHA-producing microorganism is not particularly limited, but PHAs derived from organisms belonging to the genus Ralstonia, Capriavidus, Woutersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium. Examples include synthase genes and variants thereof. As the variant, a base sequence encoding a PHA synthase in which one or more amino acid residues have been deleted, added, inserted, or substituted can be used.
  • a gene having a nucleotide sequence encoding a polypeptide represented by an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, and an amino acid sequence having a sequence identity of 85% or more to the amino acid sequence and a gene having a base sequence encoding a polypeptide having PHA synthase activity is preferably 90% or higher, more preferably 95% or higher, still more preferably 97% or higher, and particularly preferably 99% or higher.
  • the step of culturing the PHA-producing microorganism can be carried out by a person skilled in the art based on common technical knowledge, and the medium composition, carbon source addition method, culture scale, aeration and stirring conditions, culture temperature, culture time, etc. are particularly limited. not.
  • PHA can be accumulated in the cells of the PHA-producing microorganism.
  • PHA-producing microorganisms that have accumulated PHA in their cells are also referred to as PHA-accumulating cells.
  • the shape of PHA-accumulating cells which are non-spherical, preferably rod-shaped, can be confirmed by microscopic observation.
  • the shape of PHA-accumulating cells is defined by the number-average aspect ratio of the cell diameter of PHA-accumulating cells observed with a microscope.
  • the aspect ratio is the ratio (Lb/La) of the shortest minor axis length (La) and the longest major axis length (Lb) of the straight line passing through the center of the PHA-accumulating cell.
  • the number average aspect ratio is the number average value of the aspect ratios.
  • the number average aspect ratio of the cell diameter of PHA-accumulating cells is preferably 2.0 or more, more preferably 2.5 or more.
  • the upper limit of the number-average aspect ratio of the cell diameter is not particularly limited, it may be, for example, 10 or less, or 5 or less.
  • the method for calculating the number-average aspect ratio of the cell diameter of PHA-accumulating cells is as follows. In the image observed with an optical microscope, for each of 50 or more PHA-accumulating cells, the shortest short axis length (La) and the longest long axis length (Lb ) to calculate the aspect ratio (Lb/La). Then, by calculating the number average value of the obtained aspect ratios, the number average aspect ratio of the cell diameter of the PHA-accumulating cells can be obtained.
  • PHA particles are usually contained within a PHA-accumulating cell.
  • the number of PHA particles in the PHA-accumulating cells according to this embodiment is not particularly limited. However, if the total amount of PHA is the same, the larger the PHA particle size, the more fluid the aqueous suspension becomes. preferably.
  • Means for aggregating PHA particles in PHA-accumulating cells include heat or alkali treatment as disclosed in Appl Microbial Biotechnol (2019 Feb; 103(4): 1905-1917.). In particular, a method of treating with heat is preferred.
  • the heat treatment conditions are not particularly limited, but the treatment is preferably such that the PHA-accumulating cells are held at 50 to 90° C. for about 10 minutes to 7 hours.
  • a step of treating PHA-accumulating cells satisfying the above-mentioned number-average aspect ratio of cell diameter with an enzyme is carried out.
  • the enzyme it is preferable to use at least a cell wall degrading enzyme, and it is more preferable to use a protease in addition to the cell wall degrading enzyme.
  • the cell wall degrading enzyme is not particularly limited as long as it degrades the cell wall of bacteria, but examples include lysozyme, amylase, cellulase, maltase, saccharase, ⁇ and ⁇ -glycosinase. Lysozyme is particularly preferred.
  • cell wall-degrading enzymes examples include “egg white lysozyme” (manufactured by Nagase Chemtex Co., Ltd.), “lysozyme” (manufactured by Shandong Huayuan Jingmao), “biozyme A”, and “cellulase A “Amano” 3". , “Cellulase T “Amano” 4”, “ ⁇ -Glucosidase “Amano”” (manufactured by Amano Enzymes), “Termamyl”, “Cellsoft” (manufactured by Novozymes) and the like.
  • an enzyme composition containing a stabilizing agent for the enzyme, a surfactant, an anti-redeposition agent, and the like may also be used.
  • protease examples include, but are not limited to, alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase, and the like.
  • proteolytic enzymes examples include “Protease A”, “Protease P”, “Protease N” (manufactured by Amano Enzyme), “Alcalase”, “Esperase”, “Zavinase”, and “Evarase”. (above, manufactured by Novozymes) and the like. These commercial products can be suitably used from the viewpoint of decomposition activity.
  • an enzyme composition containing a stabilizing agent for the enzyme, a surfactant, an anti-soil redeposition agent, etc. may also be used.
  • the temperature and pH conditions for enzyme treatment are preferably within the optimum range of the enzyme used.
  • Alcalase manufactured by Novozymes
  • the enzyme treatment is preferably carried out at a temperature of 50-60°C and a pH of 8-9.
  • Esperase manufactured by Novozymes
  • egg white lysozyme manufactured by Nagase ChemteX Corporation
  • it is preferable to carry out enzymatic treatment at a temperature of 40-50°C and a pH of 6-7.
  • the enzymatic treatment time can be set as appropriate in consideration of the effects of the invention, and is not particularly limited, but may be, for example, within the range of 0.5 to 8 hours.
  • the amount of enzyme used is not particularly limited, and may be set appropriately in consideration of the effects of the invention depending on the type and activity of the enzyme used. It is preferably in the range of 0.001 to 10 parts by weight, more preferably 0.001 to 5 parts by weight.
  • a cell wall-degrading enzyme as an enzyme. Sphericalization is possible.
  • a cell wall-degrading enzyme and a proteolytic enzyme are used in combination, one of the enzymes may be used for enzymatic treatment and then the other enzyme may be used for enzymatic treatment, or both enzymes may be used simultaneously for enzymatic treatment. processing may be performed.
  • the cell wall-degrading enzyme itself is a protein and can be degraded by the protease, it is necessary to perform the enzymatic treatment using the cell wall-degrading enzyme, and then add the protease to perform the enzymatic treatment with the protease. is preferred.
  • Cell crushing step and PHA recovery step After the enzymatic treatment, a crushing step of crushing the microbial cells to obtain a cell lysate and a recovery step of recovering the PHA particles from the cell lysate can be performed by well-known methods.
  • the PHA recovery step may be performed directly after the enzyme treatment. It is preferable to implement the PHA recovery process after the crushing process.
  • a known method can be applied, and is not particularly limited. method, a method using a surfactant, an alkali, or the like. By carrying out such a crushing step, the PHA-accumulating cells are further crushed, and a cell lysate in which cell components other than PHA are dissolved in water can be obtained.
  • PHA particles can be recovered as an aqueous PHA suspension in which the components are diluted.
  • the PHA aqueous suspension can be used as it is for processing.
  • PHA particles can also be recovered as powder by separating the PHA particles from the PHA aqueous suspension by filtration or centrifugation and drying.
  • the PHA particles are spherical, the fluidity of the aqueous suspension is relatively good even when the PHA particles are contained at a high concentration, and the concentration step or drying step described above can be efficiently performed. be able to.
  • the spheroidization of PHA particles by the enzyme treatment described above can be evaluated by the particle diameter span value obtained from the measurement results of a laser diffraction particle size distribution meter. Specifically, the degree of decrease in the particle diameter span value of PHA obtained by crushing PHA-accumulating cells after enzyme treatment compared to the particle diameter span value of PHA obtained by crushing PHA-accumulating cells before enzyme treatment. , the spheronization of PHA particles by enzymatic treatment can be evaluated.
  • the particle size span value of PHA after enzyme treatment is preferably 10% or more smaller than the particle size span value of PHA before enzyme treatment, and is preferably 15% or more. is more preferred. In a specific formula, it is calculated by 100 ⁇ (particle size span value of PHA before enzyme treatment ⁇ particle size span value of PHA after enzyme treatment)/(particle size span value of PHA before enzyme treatment). The value is preferably 10% or more, more preferably 15% or more. Although the upper limit is not particularly limited, it may be 70% or less, or 50% or less.
  • the particle size span value indicates the particle size distribution width, and indicates the value defined by the formula (D90-D10)/D50.
  • D10, D50, and D90 represent the particle diameters corresponding to the integrated values of the particle diameter distribution of 10%, 50%, and 90%, respectively.
  • the principle of evaluating changes in the shape of PHA particles based on changes in the particle size span value uses the properties of a laser diffraction particle size distribution analyzer.
  • a laser diffraction particle size distribution meter is premised on measuring spherical particles, when measuring non-spherical particles, the particle size distribution width tends to be larger than the actual particle size distribution width.
  • the particle size distribution width naturally changes depending on the variation in particle volume. Therefore, the particle size distribution width in a laser diffraction type particle size distribution analyzer depends on the variation in volume of the particles to be measured and the shape of the particles. That is, the smaller the variation in the volume of the particles and the closer the shape of the particles to a sphere, the smaller the particle diameter span value.
  • the absolute value of the particle size span of the PHA after the enzyme treatment cannot be univocally defined because the variation in the volume of the PHA particles accumulated in the microbial cells varies depending on the type of microorganism used and the method of culturing the microorganism. Although difficult, it is usually preferably 0.85 or less, more preferably 0.70 or less. Although the lower limit of the absolute value of the grain span is not particularly limited, it may be, for example, 0.3 or more, 0.4 or more, or 0.5 or more.
  • more spherical PHA particles can be recovered from rod-shaped microbial cells that accumulate PHA.
  • the step of concentrating or drying the PHA aqueous suspension can be carried out easily and efficiently.
  • the production method according to item 9, wherein the microorganism is a transformed microorganism of Escherichia coli.
  • Method of manufacture as described.
  • [Item 13] 13 The production method according to any one of items 1 to 12, wherein the polyhydroxyalkanoic acid is a polymer containing 3-hydroxybutyric acid as a monomer unit.
  • the polyhydroxyalkanoic acid is a copolymer of two or more hydroxyalkanoic acids.
  • a plasmid vector pCUP2-REP-phaCAB for expressing the phaCAB gene was constructed.
  • the gene expression plasmid vector pCUP2-REP-phaCAB was introduced into Escherichia coli BW25113 strain by the heat shock method.
  • KNK-005 strain was cultured under the following conditions to accumulate PHA in the cells, and then the shapes of the cells and PHA particles were evaluated.
  • the KNK-005 strain is a transformant in which a PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence of SEQ ID NO: 3) is introduced onto the chromosome of the Capriavidus necator H16 strain. and can be produced according to the method described in US Pat. No. 7,384,766.
  • composition of the seed medium is 1 w/v % Meat-extract, 1 w/v % Bacto-Tryptone, 0.2 w/v % Yeast-extract, 0.9 w/v % Na 2 HPO 4 ⁇ 12H 2 O, 0.15 w. /v% KH2PO4 , (pH 6.8).
  • the composition of the pre-culture medium is 1.1 w/v% Na 2 HPO 4 .12H 2 O, 0.19 w/v% KH 2 PO 4 , 1.29 w/v% (NH 4 ) 2 SO 4 , 0.1 w.
  • Palm olein oil was added all at once at a concentration of 10 g/L as a carbon source.
  • the composition of the PHA production medium is 0.385 w/v% Na2HPO4.12H2O , 0.067 w/v% KH2PO4 , 0.291 w/v% ( NH4 ) 2SO4 , 0.1 w/v% v% MgSO4.7H2O , 0.5v/v% trace metal salt solution ( 1.6w/ v % FeCl3.6H2O , 1w/v% CaCl2.2H2O , 0.5v/v% in 0.1N hydrochloric acid ) .02 w/v% CoCl 2 .6H 2 O, 0.016 w/v% CuSO 4 .5H 2 O, and 0.012 w/v% NiCl 2 .6H 2 O).
  • the ratio of accumulated PHA to dry cells (hereinafter also referred to as PHA content) was measured as follows. Cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain dried cells. 100 ml of chloroform was added to 1 g of the obtained dried cells, and the mixture was stirred at room temperature for a whole day and night to extract PHA in the cells. After removing the bacterial cell residue by filtration, it was concentrated with an evaporator until the total volume reached 30 ml, and then 90 ml of hexane was gradually added and allowed to stand for 1 hour while slowly stirring. After the precipitated PHA was separated by filtration, it was vacuum-dried at 50° C. for 3 hours. The weight of the dry PHA was measured, and the ratio of the dry PHA weight to the dry cell weight was calculated as the PHA content.
  • the PHA particle size span was measured as follows. After the bacterial cell inactivation treatment described later, the culture solution before the enzyme treatment or after the enzyme treatment was suspended in an aqueous sodium dodecyl sulfate solution with a final concentration of 3.3%, and the cells were crushed by ultrasonication.
  • the particle size (D10, D50, D90) of the PHA particles was measured by analysis using a laser diffraction/scattering particle size distribution analyzer (Microtrac MT3300EXII manufactured by Microtrac Bell). Measurements were performed with standard settings (particle permeability: transparent, particle refractive index: 1.81, particle shape: aspherical, solvent refractive index: 1.333). From the measured D10, D50, and D90 values, (D90-D10)/D50 was calculated as the PHA particle size span.
  • PHA production culture PHA production culture was performed as follows. First, the KNK-005 strain glycerol stock (50 ⁇ l) was inoculated into a seed medium (10 ml) and cultured for 24 hours to perform seed culture. Next, 1.0 v/v % of the seed culture was inoculated into a 3 L jar fermenter (MDL-300 manufactured by Marubishi Bioengineering Co., Ltd.) containing 1.8 L of preculture medium. The operating conditions were culture temperature of 33° C., agitation rate of 500 rpm, aeration rate of 1.8 L/min, and pH was controlled between 6.7 and 6.8 for 28 hours of culture for pre-culture. A 14% ammonium hydroxide aqueous solution was used for pH control.
  • 5.0 v/v % of the preculture solution was inoculated into a 5 L jar fermenter (MDS-U50 model manufactured by Marubishi Bioengineering Co., Ltd.) containing 2.5 L of PHA production medium.
  • the operating conditions were culture temperature of 33° C., stirring speed of 420 rpm, aeration rate of 2.1 L/min, and pH was controlled between 6.7 and 6.8.
  • a 25% ammonium hydroxide aqueous solution was used for pH control.
  • the carbon source was added intermittently. Palm olein oil was used as the carbon source. Culture was continued until the PHA content reached about 90%. After completion of the culture, the culture solution was heat-treated at 70° C.
  • FIG. 1 shows a photograph taken during the observation of the cells with an optical microscope as described above.
  • the KNK-005 strain had a number-average aspect ratio of the cell diameter after PHA accumulation of 1.55 and a spherical cell shape.
  • the minCD gene derived from Capriavidus necator (the gene encoding the cell division inhibitory enzyme described in SEQ ID NO: 7) was introduced onto the chromosome of the KNK-005 strain, and the A2405 gene (SEQ ID NO: 8 gene encoding the cell wall-degrading enzyme described in 1) is disrupted, and can be produced according to the method described in WO2021/049207.
  • the number average aspect ratio of cell diameter, and PHA particle size span were measured as described above. Table 1 shows the results.
  • FIG. 2 shows microscopic photographs of the cells observed as described above.
  • the number average aspect ratio of the cell diameter after PHA accumulation was 3.51, and the cell shape was rod-shaped.
  • the PHA particle size span was 46% or more larger.
  • Example 1 Shape evaluation of PHA particles of minCD-expressing A2405-disrupted strain after cell wall-degrading enzyme treatment After cooling the culture solution after the bacterial cell inactivation treatment in Comparative Example 1 to 50°C, egg white, which is a cell wall-degrading enzyme, Lysozyme (manufactured by Nagase Chemtex Co., Ltd.) was added in an amount corresponding to 0.0036% by weight of the bacterial cell components contained in the culture medium, and the mixture was stirred for 2 hours while controlling the pH at 6-7. PHA particle size span was measured as described above. Table 1 shows the results.
  • Example 1 compared with Comparative Example 1, the PHA particle size span was reduced by 13% or more, indicating that the PHA particles were sphericalized by the treatment with the cell wall-degrading enzyme.
  • Example 2 Shape evaluation of PHA particles of minCD-expressing A2405-disrupted strain after cell wall-degrading enzyme treatment and proteolytic enzyme treatment Alcalase (manufactured by Novozyme) was added in an amount corresponding to 1.1% by weight of the amount of bacterial cells contained in the culture solution, and stirred for 2 hours while controlling the pH at 8.2 to 8.8. PHA particle size span was measured as described above. Table 1 shows the results.
  • Example 2 compared with Comparative Example 1, the PHA particle diameter span was reduced by 32% or more, and the PHA particles were further sphericalized by the treatment using the cell wall-degrading enzyme and the proteolytic enzyme. I understand.
  • composition of the pre-culture medium was 1 w/v% Bacto-Tryptone, 0.5 w/v% Yeast-extract, and 1 w/v% NaCl12H 2 O (pH 6.8).
  • the composition of the PHA production medium was 1 w/v% Bacto-Tryptone, 0.5 w/v% Yeast-extract, 1 w/v% NaCl12H 2 O (pH 6.8), and 2% Glucose.
  • PHA production culture PHA production culture was performed as follows. First, a glycerol stock (50 ⁇ l) of the BW25113 phaCAB expressing strain was inoculated into a preculture medium (5 ml) and cultured at 37° C. for 18 hours to perform seed culture. Next, 1.0 v/v % of the preculture was inoculated into a 500 mL Sakaguchi flask containing 100 mL of PHA production medium. Culturing was performed at a culture temperature of 37° C. and a shaking speed of 120 rpm. Culture was continued until the PHA content reached about 60%. After completion of the culture, the culture solution was heat-treated at 60° C.
  • FIG. 3 shows a photograph taken during microscopic observation of the cells as described above.
  • the number average aspect ratio of the cell diameter after PHA accumulation was 3.02, and the cell shape was rod-shaped.
  • Example 3 Shape evaluation of PHA particles of BW25113 phaCAB expressing strain after treatment with cell wall-degrading enzyme After cooling the culture solution after the bacterial cell inactivation treatment in Comparative Example 2 to 50°C, egg white lysozyme, which is a cell wall-degrading enzyme, was added. (manufactured by Nagase Chemtex Co., Ltd.) was added in an amount corresponding to 0.0036% by weight of the amount of bacterial cells contained in the culture solution, and the mixture was stirred for 2 hours while controlling the pH at 6-7. PHA particle size span was measured as described above. Table 1 shows the results.
  • Example 3 compared with Comparative Example 2, the PHA particle size span was reduced by 11% or more, indicating that the PHA particles were spheroidized by the treatment using the cell wall-degrading enzyme.
  • Example 4 Shape evaluation of BW25113 phaCAB-expressing strain cells and PHA particles after cell wall degrading enzyme treatment and proteolytic enzyme treatment Alcalase (manufactured by Novozyme), an enzyme, was added in an amount corresponding to 1.1% by weight of the bacterial cell component contained in the culture solution, and the mixture was stirred for 2 hours while controlling the pH at 8.2 to 8.8. . PHA particle size span was measured as described above. Table 1 shows the results.
  • Example 4 compared with Comparative Example 2, the PHA particle diameter span was reduced by 17% or more, and the PHA particles were further sphericalized by the treatment with the cell wall-degrading enzyme and the proteolytic enzyme. I understand.

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JP2001226567A (ja) * 2000-02-18 2001-08-21 Canon Inc 糖鎖高分子組成物、その製造方法及び成形体
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