WO2015133468A1 - Procédé de production d'un copolymère de polyester à l'aide d'une souche recombinante présentant un locus modifié pour la protéine qui permet la liaison aux granules de polyester - Google Patents

Procédé de production d'un copolymère de polyester à l'aide d'une souche recombinante présentant un locus modifié pour la protéine qui permet la liaison aux granules de polyester Download PDF

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WO2015133468A1
WO2015133468A1 PCT/JP2015/056201 JP2015056201W WO2015133468A1 WO 2015133468 A1 WO2015133468 A1 WO 2015133468A1 JP 2015056201 W JP2015056201 W JP 2015056201W WO 2015133468 A1 WO2015133468 A1 WO 2015133468A1
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pha
nucleic acid
seq
strain
gene
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Japanese (ja)
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俊昭 福居
和泉 折田
由依 川島
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Kaneka Corp
Tokyo Institute of Technology NUC
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Kaneka Corp
Tokyo Institute of Technology NUC
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the present invention is based on poly (3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid), which is one of copolymerized polyhydroxyalkanoic acids, which can be decomposed by microorganisms using vegetable oil as a basic raw material and has excellent biocompatibility. ) By a microorganism and controlling the composition ratio of the monomer units in the copolymer.
  • Petroleum synthetic plastic an essential material in modern society, is cheap and easy to process, but it is difficult to dispose of it because of its indegradability.
  • Polyhydroxyalkanoic acid (hereinafter abbreviated as “PHA”) that accumulates in cells as an energy source for microorganisms is expected to be a plastic material of environmentally low load type that uses renewable biomass as a raw material and has biodegradability.
  • PHA Polyhydroxyalkanoic acid
  • P (3HB) Poly ((R) -3-hydroxybutanoic acid)
  • P (3HB) is a typical PHA biosynthesized by many microorganisms, but is practical because of its physical properties of being hard and brittle. Is difficult.
  • Aeromonas caviae isolated from soil is obtained from (R) -3-hydroxybutanoic acid using (R) -3-hydroxybutanoic acid as a raw material.
  • Poly ((R) -3-hydroxybutanoic acid-co- (R) -3-hydroxyhexanoic acid) copolymer which is a copolyester composed of R) -3-hydroxyhexanoic acid hereinafter abbreviated as 3HHx
  • 3HHx a copolyester composed of R) -3-hydroxyhexanoic acid
  • Patent Document 1 P (3HB-co-3HHx) biosynthesized by Caviae from vegetable oil has a 3HHx fraction of 10 to 20 mol%, showing flexibility suitable for practical use, but its cell accumulation rate is as low as about 15% by weight. It was difficult to apply to actual production (Patent Document 1, Patent Document 2, Non-Patent Document 1).
  • Non-Patent Literature 4 Necrotor strain chromosome Caviae-derived PHA synthase gene introduction
  • Patent Literature 5 inactivation of 3HB monomer supply enzyme ⁇ -ketothiolase gene or acetoacetyl-CoA reductase
  • Patent Literature 5 R-form-specific enoyl-CoA hydratase gene (phaJ) which converts 2-enoyl-CoA, an intermediate in the fatty acid ⁇ -oxidation pathway, to (R) -3-hydroxyacyl-CoA ((R) -3HA-CoA) )
  • Patent document 6 non-patent document 3, non-patent document 4
  • modification by gene disruption of ⁇ -oxidation pathway enzyme non-patent document 5
  • PHA biosynthesized in cells exists as water-insoluble granules in the cells, and its surface is covered with a lipid monolayer, and is a granule-binding protein called phasin, PHA polymerizing enzyme, and PHA which is a degrading enzyme. Depolymerase is bound.
  • the granule-binding protein is an amphipathic protein, and it has been proposed to maintain the granule in the cytoplasm by binding to the highly hydrophobic polyester granule surface and to adjust the ratio of the surface area to the granule volume. Yes. More recently, granule-binding proteins have been found to interact with PHA polymerizing enzymes (Wieczorek, R.
  • the present inventors have found that an excellent production strain capable of biosynthesizing P (3HB-co-3HHx) having a high 3HHx fraction from a vegetable oil raw material at a high accumulation rate can be provided, and the present invention has been completed.
  • the present invention is as follows.
  • a recombinant strain imparted with the ability to produce copolymerized PHA introduces one or more genes encoding a broad substrate-specific PHA polymerase and one or more R-isomer-specific enoyl-CoA hydratase genes The method according to [1] above, wherein the strain is a cultured strain.
  • the copolymerized PHA is poly (3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid), and increases the fraction of 3-hydroxyhexanoic acid in the copolymerized PHA. 2].
  • the gene encoding the PHA granule binding protein used for replacement is (A) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 2; or (b) an activity that hybridizes with a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and binds to PHA granules.
  • the method according to [1] to [5] above which comprises a nucleic acid encoding a protein having [7]
  • the R-form-specific enoyl-CoA hydratase gene is (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3; or (b) hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 under stringent conditions and in the middle of the fatty acid ⁇ -oxidation system.
  • the method according to any one of [1] to [6] above comprising a nucleic acid encoding a protein having an activity of converting the body into (R) -3-hydroxyacyl-CoA.
  • One of the genes encoding a broad substrate-specific PHA synthase is (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1; or (b) an activity of hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and synthesizing PHA.
  • One of the genes encoding PHA granule binding proteins used for replacement is (A) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 2; or (b) an activity that hybridizes with a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and binds to PHA granules.
  • a R-form specific enoyl-CoA hydratase gene (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3; or (b) hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 under stringent conditions and in the middle of the fatty acid ⁇ -oxidation system.
  • the method according to any one of [1] to [7] above, comprising a nucleic acid encoding a protein having an activity of converting the body into (R) -3-hydroxyacyl-CoA.
  • the present invention provides a recombinant strain imparted with the ability to produce copolymer PHA by introducing a gene encoding one or more broad substrate-specific PHA synthases into a host different from the species of the gene.
  • a monomer in a copolymerized PHA comprising replacing a gene encoding a main PHA granule-binding protein in the recombinant strain with a gene encoding a PHA granule-binding protein derived from the same species as the PHA-polymerizing enzyme
  • the present invention relates to a method for controlling the composition ratio of units.
  • the host used in the present invention is not particularly limited as long as it is different from the species of PHA synthase gene (phaC) introduced into the host.
  • necator strain A. Caviae strain and Escherichia coli Escherichia coli strain can be mentioned.
  • the phaC to be introduced is A.I.
  • the host may be any other microbial species (for example, a C. necator strain). According to the present invention, it is possible to use a recombinant strain in which a gene encoding a broad substrate-specific PHA synthase described later is introduced in advance into a host different from the species of the gene.
  • the R-form-specific enoyl-CoA hydratase gene described later may be further introduced into the recombinant strain.
  • the foreign species encoding the broad substrate-specific PHA polymerizing enzyme and the species of the gene encoding the PHA granule-binding protein described later are: The same, but preferably different from the species of the recombinant strain.
  • a foreign gene encoding a broad substrate-specific PHA synthase foreign phaC
  • a foreign gene encoding one or more broad substrate specific PHA polymerases (hereinafter abbreviated as foreign phaC) is used in the host.
  • foreign phaC broad substrate specific PHA polymerases
  • the “broad substrate-specific PHA polymerization enzyme” used for controlling the copolymer composition of the present invention is an enzyme whose polymerization activity for synthesizing PHA is not limited to only one type of (R) -3HA-CoA. Say.
  • PHA polymerase with broader substrate specificity can be used for microorganisms in which biosynthesis of copolymerized PHA is not possible due to the substrate specificity of PHA polymerase or the composition of the copolymer is limited.
  • PHA in which various 3-hydroxyalkanoic acid units are copolymerized can be synthesized.
  • a recombinant strain into which a foreign gene of a PHA synthase having activity against (R) -3HA-CoA having 4 to 6 carbon atoms has been introduced may have P ( 3HB-co-3HHx) has been reported (Patent Literature 3, Patent Literature 4, Non Patent Literature 2, and Non Patent Literature 3).
  • Examples of strains expressing such a broad substrate-specific PHA synthase include A. Caviae FA440 strain (FERM P-15786) (Patent No. 3062459), Aeromonas hydrophila WQ strain and 4AK5 strain (Lu, X. et al., FEMS Microbiol. Lett., 243: 149-55 (2005)). Etc. are known.
  • the exogenous phaC used in the present invention includes single-stranded or double-stranded DNA and its RNA complement.
  • DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the nucleic acid used in the present invention DNA is preferable.
  • codons are degenerate and some amino acids have multiple base sequences encoding one amino acid.
  • Nucleic acids having any base sequence are within the scope of the present invention.
  • the number of exogenous phaC introduced into the host may be one or more. Specifically, the number of genes introduced is one, two, three, four, or five, preferably one or two.
  • the gene encoding a broad substrate-specific PHA synthase used for copolymer composition control is preferably phaC NSDG .
  • phaC NSDG refers to A.I. It is a gene that encodes a mutant (NSDG mutant) in which the asparagine at position 149 of serine and the aspartic acid at position 171 are replaced by glycine in the polyhydroxyalkanoic acid synthase derived from Caviae strain.
  • the cloning of the phaC NSDG gene can be performed by an ordinary molecular biological technique. For example, Tsuge, T .; Et al., FEMS Microbiol. Lett. 277: 217-222 (2007); Kichise, T .; Et al., Appl. Environ. Microbiol. 68: 2411-2419 (2002); JP 2008-29218; International Publication WO2011 / 105369.
  • the phaC NSDG gene comprises (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 and stringent conditions It may consist of a nucleic acid that encodes a protein that hybridizes underneath and has an activity to synthesize PHA.
  • the phaC NSDG gene comprises (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 and a string. It may be composed of a nucleic acid that encodes a protein that hybridizes under gentle conditions and has an activity to synthesize PHA.
  • the phaC NSDG gene is a synthetic nucleotide designed as a primer based on the nucleotide sequence of SEQ ID NO: 1, as described in Example 1 described later.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • transcription-based amplification Kwoh DY et al., Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)
  • SDA strand displacement reactions
  • under stringent conditions means to hybridize under moderate or high stringent conditions.
  • moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of DNA.
  • Basic conditions are described in Sambrook, J. et al. Are shown in Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001), with regard to nitrocellulose filters, 5 ⁇ SSC, 0.5% SDS, 1.
  • High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. Generally, such conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions, such as hybridization conditions as described above, and about 68 ° C. Defined with 0.2 ⁇ SSC, 0.1% SDS wash.
  • the temperature and wash solution salt concentration can be adjusted as needed according to factors such as the length of the probe.
  • Homologous nucleic acids cloned using the nucleic acid amplification reaction or hybridization as described above are at least 30% or more, preferably 50% or more, respectively, more preferably than the base sequence described in SEQ ID NO: 1. Has an identity of 70% or more, even more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. Note that the percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by Devereux et al., Nucl. Acids Res. , 12, 387 (1984) and determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3) available from the University of Wisconsin Genetics Computer Group (UWGCG). can do.
  • GAP computer program GAP computer program
  • the gene encoding a broad substrate-specific PHA synthase is one or two phaC NSDG genes, and may be a nucleic acid comprising the base sequence represented by SEQ ID NO: 1. Good.
  • genes introduced on the chromosome of the host are appropriately transcribed and further translated into a protein having the desired activity, these genes are suitable on the chromosome. It needs to be integrated so that it is under the control of the promoter.
  • PHA granule binding protein (phaP)
  • the main PHA granule-binding protein is converted into the PHA.
  • PHA granule-binding protein used in the present invention means a protein having an activity of binding to the surface of PHA granules accumulated in cells. PhaP1 (Wieczorek, R.
  • PhaP2 PhaP3, PhaP4 (Potter, M. et al., Microbiology, 151, 825-833) (2005)
  • PhaP in Caviae FA440 strain (Fukui, T. et al., Biomacromolecules, 2, 148-153 (2001)
  • PhaP in Paracoccus denitificicans strain (Maehara, A. et al., J. Bacteriol., 181, 294-21-29) Etc. are known.
  • “major” PHA granule-binding protein means that when a plurality of PHA granule-binding proteins are present in the cell, it is present in the largest amount.
  • PhaP1 corresponds (Wieczorek, R. et al., J. Bacteriol., 177, 2425-2435 (1995)).
  • the exogenous phaC encoding a broad substrate-specific PHA synthase introduced into the host and the gene of the main PHA granule binding protein functioning in the recombinant strain are the same organism.
  • the phaP present in the recombinant strain imparted with the ability to produce copolymerized PHA as a host is different from the species of foreign phaP introduced for composition control.
  • the origin of exogenous phaC encoding a broad substrate-specific PHA synthase is In the case of a Caviae strain, the origin of the foreign phaP introduced into the host is A.
  • An embodiment that is a Caviae strain is included in the present invention.
  • the foreign phaC and foreign phaP introduced into the host are not particularly limited as long as they are the same species.
  • the phaP used in the present invention includes single-stranded or double-stranded DNA and its RNA complement.
  • DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the nucleic acid used in the present invention DNA is preferable. As is well known, codons are degenerate and some amino acids have multiple base sequences that encode one amino acid. However, if the base sequence of a nucleic acid that encodes a protein having activity to bind to PHA granules, A nucleic acid having any base sequence is also included in the scope of the present invention.
  • a gene (phaP) encoding a PHA granule-binding protein used for copolymer composition control is A.I. from Caviae strains or C.I. It is preferably derived from a necator strain.
  • “phaP Ac ” refers to A.I. It refers to a gene encoding a PHA granule binding protein derived from a Caviae strain.
  • GenBank GenBank Accession No. Nucleotides 2197-2547 (“ORF1”) in D88825 can be utilized. In this specification, the base sequence of the gene is described in the sequence listing as SEQ ID NO: 2.
  • phaP1 Cn refers to C.I. It refers to the gene encoding the major PHA granule binding protein derived from the necator strain.
  • C.I. Necator genome NC No. 01576.16.1
  • the nucleotide sequence of the gene is described in the sequence listing as SEQ ID NO: 4.
  • the phaP Ac gene and the phaP1 Cn gene can be isolated and identified by ordinary molecular biological techniques.
  • the exogenous phaP introduced into the host is (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2; It may consist of a nucleic acid that encodes a protein that hybridizes under stringent conditions and has an activity of binding to PHA granules.
  • the exogenous phaP is (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2 and a stringent It may consist of a nucleic acid that encodes a protein that hybridizes under conditions and has an activity of binding to PHA granules.
  • the phaP to be substituted present on the host chromosome is (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 4; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 4. It may be a thing.
  • the “stringent conditions” are as described above, and the homologous nucleic acid cloned using a nucleic acid amplification reaction, hybridization, or the like, respectively, with respect to the base sequence described in SEQ ID NO: 2, At least 30% or more, preferably 50% or more, more preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more.
  • the percent identity can be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences can be determined by Devereux et al., Nucl. Acids Res. , 12, 387 (1984) and determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3) available from the University of Wisconsin Genetics Computer Group (UWGCG). can do.
  • the exogenous phaP introduced into the host may be phaP Ac and may be a nucleic acid comprising the base sequence represented by SEQ ID NO: 2.
  • SEQ ID NO: 2 the base sequence represented by SEQ ID NO: 2.
  • the “R-form-specific enoyl-CoA hydratase” used in the present invention is an enzyme that converts enoyl-CoA, which is a fatty acid ⁇ -oxidation intermediate, into (R) -3HA-CoA, which is a PHA monomer.
  • the origin of the biological species is not particularly limited.
  • phaJ used in the present invention includes single-stranded or double-stranded DNA and its RNA complement.
  • DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the nucleic acid used in the present invention DNA is preferable.
  • codons are degenerate and some amino acids have a plurality of base sequences encoding one amino acid.
  • any base sequence of a nucleic acid encoding R-isomer-specific enoyl-CoA hydratase can be used. Nucleic acids having the base sequences are also included in the scope of the present invention.
  • phaJ used in the present invention
  • A. Base sequence of R-specific enoyl-CoA hydratase gene (phaJ Ac ) derived from Caviae strain: GenBank Accession No. Nucleotides 4475-4879 (“ORF3”) in D88825 can be utilized.
  • the number of genes encoding R-isomer specific enoyl-CoA hydratase may be one or more. Specifically, the number of genes introduced is one, two, three, four, or five, preferably one or two.
  • phaJ introduced into the host is A.
  • Caviae-derived phaJ Ac is preferable. Isolation and identification of phaJ Ac can be performed by an ordinary molecular biological technique.
  • Patent Document 3 (described above) may be referred to.
  • phaJ is (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3; or (b) a stringent nucleic acid comprising the nucleic acid comprising the base sequence represented by SEQ ID NO: 3.
  • phaJ is (a) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3; or (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and stringent conditions It may be composed of a nucleic acid that encodes a protein that hybridizes below and has an activity of converting a fatty acid ⁇ -oxidation intermediate into (R) -3-hydroxyacyl-CoA.
  • the “stringent conditions” are as described above, and the homologous nucleic acid cloned using a nucleic acid amplification reaction, hybridization, or the like, respectively, with respect to the base sequence described in SEQ ID NO: 3, At least 30% or more, preferably 50% or more, more preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more.
  • the percent identity can be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences can be determined by Devereux et al., Nucl. Acids Res. , 12, 387 (1984) and determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3) available from the University of Wisconsin Genetics Computer Group (UWGCG). can do.
  • the gene encoding R-specific enoyl-CoA hydratase introduced into the host may be phaJ Ac and may be a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3.
  • these genes are under the control of appropriate promoters in order for the genes introduced into the host to be properly transcribed and further translated into proteins with the desired activity. Need to be incorporated.
  • the method for introducing foreign phaC, foreign phaP having the same origin as foreign phaC, and phaJ into the host is not particularly limited, and may be introduction using a plasmid vector capable of autonomous replication in the host. It may be introduced into the chromosome by replacement.
  • exogenous phaP may be introduced by recombination, or exogenous phaP may be introduced by a plasmid vector capable of autonomous replication into a strain in which the function of endogenous phaP has been lost by mutation, destruction, or deletion.
  • exogenous phaP may be introduced by a plasmid vector capable of autonomous replication into a strain in which the function of endogenous phaP has been lost by mutation, destruction, or deletion.
  • these genes need to be incorporated under the control of an appropriate promoter.
  • each gene to be introduced is not limited as long as the desired effect is achieved.
  • the positional relationship of each introduced gene is not particularly limited, and may be arranged on or separated from each other on a chromosome or in an autonomously replicating vector, and a part thereof is introduced into the chromosome by homologous recombination. Then, the rest may be introduced with an autonomously replicating vector, or introduced with a plurality of different compatible autonomously replicating vectors.
  • a strain in which pphaC NSDG is pre-integrated on a chromosome may be used.
  • An NSDG strain is exemplified as such a microorganism (see Patent Document 6).
  • “NSDG strain” means C.I. necator, H16 strain (ATCC16699 strain, DSM428 strain) which is a kind of wild strain
  • the original phaC in the pha operon on the chromosome of This is a recombinant strain in which phaC NSDG , which is a gene of Caviae-derived PHA synthase mutant enzyme, is substituted by homologous recombination.
  • a strain in which pphaC NSDG and phaJ are pre-integrated on a chromosome may be used as long as the object of the present invention is achieved.
  • An example of such a microorganism is the MF02 strain (see Patent Document 6).
  • the MF02 strain is a recombinant strain in which phaJ Ac is integrated between phaC NSDG and phaA ( ⁇ -ketothiolase gene) in the pha operon on the chromosome of NSDG strain.
  • a vector can be prepared simply by ligating a desired gene to a recombination vector (for example, plasmid DNA) available in the technical field by a conventional method.
  • a recombination vector for example, plasmid DNA
  • a polyester-polymerizing enzyme gene derived from a microorganism already incorporated in the chromosome of a microorganism is used as a foreign substrate-specific polyester polymerizing enzyme.
  • a recombinant vector suitable for the host cell in order to express the desired protein can do.
  • a vector is a region in which the gene used in the present invention functions so as to cause homologous recombination with the gene of the target host cell (if necessary, an autonomous replication origin, a junction transmission region, a selection marker (for example, , Kanamycin resistance gene), etc.) are appropriately arranged or introduced so that the nucleic acid is constructed or constructed so that it is appropriately recombined.
  • a transformant can be prepared by incorporating a recombinant vector into a host cell.
  • the host cell can be either a prokaryotic cell (for example, E. coli (S17-1 strain, etc.), Bacillus subtilis) or a eukaryotic cell (mammalian cell, yeast, insect cell, etc.).
  • Introduction (transformation) of a recombinant vector into a host cell can be performed using a known method.
  • bacteria E. coli, Bacillus subtilis, etc.
  • the method of Cohen et al. Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)
  • the protoplast method Mol. Gen.
  • conjugation transfer method can be used to introduce expression vectors into cells belonging to the genus Ralstonia, Alcaligenes, Pseudomonas, etc. (J. Bacteriol., 147, 198). (1981)).
  • This conjugation transfer method is based on the nature of cells that transfer a chromosome genome or plasmid from one cell to another by contact between cells. For example, self-transmission carrying the target DNA.
  • a series of bridge formation in both cells a series of bridge formation in both cells, replication and transfer of the plasmid, and separation of the cells upon completion of DNA synthesis It is a means that enables gene transfer by a process.
  • the synthesis of copolymerized PHA involves introducing a gene encoding one or more broad substrate specific PHA polymerase into a host different from the species of the gene.
  • the gene encoding the PHA granule binding protein present on the chromosome of the recombinant strain is derived from the same species as the gene encoding the PHA polymerase.
  • copolymerized PHA is generated and accumulated in the recombinant strain or in a culture (for example, a medium), and the desired copolymerized PHA is obtained from the recombinant strain or culture. It is done by collecting.
  • the recombinant strain is preferably placed under suitable culture conditions in order to synthesize copolymerized PHA. The culture conditions of the parent strain before performing such recombinant strain culture and gene recombination may be followed.
  • the recombinant strain may be grown in a medium containing vegetable oil or fatty acid as a carbon source.
  • C.I As a medium in the case of using a necator strain as a host, vegetable oil that can be assimilated by the microorganism strain or a medium- or long-chain fatty acid having 6 or more carbon atoms was added to restrict any of nitrogen sources, inorganic salts, and other organic nutrient sources.
  • a culture medium is mentioned.
  • the medium temperature is in the range of 25 ° C. to 37 ° C., and aerobically cultured for 1 to 10 days, so that copolymerized PHA is produced and accumulated in the cells, and then recovered and purified.
  • the desired copolymerized PHA can be prepared.
  • vegetable oil when using vegetable oil as a carbon source, generally available vegetable oil can be used as vegetable oil which can be used,
  • the supply source is not specifically limited.
  • the concentration of the vegetable oil in the medium is preferably 0.1 to 5%, but can be appropriately adjusted by those skilled in the art.
  • a nitrogen source or an inorganic substance may be added to the medium.
  • the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate and the like, as well as peptone, meat extract, yeast extract, corn steep liquor and the like.
  • inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
  • Cultivation is usually carried out using shaking culture, and it is preferable to carry out the aerobic conditions at 25 ° C. to 37 ° C. for at least one day after induction of gene expression.
  • an antibiotic kanamycin, ampicillin or the like may be added to the medium.
  • arabinose, indoleacrylic acid (IAA), isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) or the like can be used as a gene expression inducer.
  • IAA indoleacrylic acid
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • a person skilled in the art can appropriately select culture conditions and conditions for inducing gene expression that are possible for desired gene expression.
  • a gene encoding one or more broad substrate specific PHA polymerases is introduced into a host different from the species of the gene.
  • the gene encoding the PHA granule binding protein present in the recombinant strain is derived from the same species as the gene encoding the PHA polymerase.
  • control means changing the composition ratio of the monomer units constituting the copolymerized PHA produced by the present invention to a desired value or range. More specifically, such a composition ratio is controlled by selecting genes introduced into the host (ie, exogenous phaC, phaP and phaJ having the same origin as the exogenous phaC), and appropriately changing the species. It can be carried out. Moreover, as one aspect, the expression of these genes introduced onto the chromosome can be performed by changing the activity of regulatory factors (eg, transcription factors, promoters, enhancers). Furthermore, mutation may be added to the gene introduced into the host to alter the function of the gene. These modifications can be performed using a general genetic engineering technique based on the genome information of the host and the base sequence information of the introduced gene. Moreover, you may change suitably the culture conditions of a recombinant strain.
  • regulatory factors eg, transcription factors, promoters, enhancers
  • the copolymer PHA is poly (3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) (hereinafter sometimes referred to as “P (3HB-co-3HHx)”)
  • the fraction of 3-hydroxyhexanoic acid (3HHx) in the copolymerized PHA can be controlled, and preferably the fraction of 3HHx can be increased, for example as detailed in Example 2 below.
  • the 3HHx fraction in P (3HB-co-3HHx) was 6.5 mol%.
  • the phaP1 Cn gene present on the chromosome of the strain by substituting phaP1 Ac gene from the same A.caviae strain as phaC NSDG gene, 3
  • the Hx percentage could be increased to 9.3mol%.
  • copolymerized PHA can be purified as follows: a transformant is recovered from the medium by centrifugation, washed with distilled water, dried or frozen. dry. Thereafter, the transformant dried in chloroform is suspended and stirred at room temperature for a predetermined time to extract copolymerized PHA. In the extraction stage, heating may be performed if necessary. The residue is removed by filtration, methanol is added to the supernatant to precipitate copolymer PHA, and the precipitate is filtered or centrifuged to remove the supernatant and dried to obtain purified copolymer PHA. Thereafter, although not limited, the composition ratio of the monomer units of the obtained copolymer PHA can be confirmed using NMR (nuclear magnetic resonance) and gas chromatography.
  • the 3HHx fraction when the copolymerized PHA is P (3HB-co-3HHx), the 3HHx fraction may be at least 1% mol or more, for example, 1 mol%, 2 mol%, 3 mol %, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, or more.
  • the 3HHx fraction may be 99 mol% or less, for example, 99 mol%, 98 mol%, 97 mol%, 96 mol%, 95 mol%, 94 mol%, 93 mol%, 92 mol%. 91 mol%, 90 mol%, or less.
  • the possible range of the 3HHx fraction is not limited, but for example, 1 to 99 mol%, 1 to 95 mol%, 1 to 90 mol%, 1 to 85 mol%, 1 to 80 mol%, 1 to 75 mol %, 1 to 70 mol%, 1 to 65 mol%, 1 to 60 mol%, 1 to 55 mol%, 1 to 50 mol%, 1 to 45 mol%, 1 to 40 mol%, 1 to 35 mol%, 1 to 30 mol%, 1 to 25 mol%, 1 to 20 mol%, 2 to 99 mol%, 2 to 95 mol%, 2 to 90 mol%, 2 to 85 mol%, 2 to 80 mol%, 2 to 75 mol%, 2 to 70 mol%, 2 to 65 mol%, 2 to 60 mol%, 2 to 55 mol%, 2 to 50 mol%, 2 to 45 mol%, 2 to 40 mol%, 2 to 35 mol%, 2 to 30 mol%, 2 to 25 mol%, 2 to 20 mol
  • the 3HHx fraction is preferably 3 to 90 mol%, more preferably 4 to 80 mol%, and still more preferably 5 to 70 mol%.
  • mol% refers to the sum of the number of moles of each component in a multi-component system divided by the number of moles of a component.
  • the copolymerized PHA obtained by the control method of the present invention is accumulated in the cells at a rate of 50 to 95% by weight, preferably 70 to 95% by weight, based on the dry cell weight.
  • Example 1 Production of Vector for Homologous Recombination
  • a series of recombinant vectors used in the present invention were produced as follows. In the following operations, unless otherwise specified, KOD Plus DNA polymerase (Toyobo) is used as a DNA polymerase for PCR, and T4 polynucleotide kinase (Toyobo) is used for 5′-phosphorylation.
  • KOD Plus DNA polymerase Toyobo
  • T4 polynucleotide kinase Toyobo
  • Bovine small intestine-derived alkaline phosphatase was used for 5'-dephosphorylation reaction
  • Ligation High Toyobo was used for ligation of DNA fragments.
  • Sequence 1 CGGGATCCCCTGGTGCACATCCAGGTCGACCACG (SEQ ID NO: 5)
  • Sequence 2 TGCTGGTCTCCAGTGGTGAACTTC (SEQ ID NO: 6)
  • Sequence 3 CGGGATCCGACGCGTTCTATGTTGCCTCTCAC (SEQ ID NO: 7)
  • Sequence 4 TAACTGCCCTGCGTTGAAGATGAC (SEQ ID NO: 8)
  • the amplified downstream fragment of phaP1 Cn was 5′-phosphorylated by kinase treatment and ligated with the upstream fragment.
  • a fragment (1.6 kb) in which the upstream and downstream of phaP1 Cn were ligated was amplified by PCR using the oligonucleotides of sequence 1 and sequence 3 as primers. PCR was performed for 30 cycles, with a reaction of 98 ° C. for 20 seconds, 59 ° C. for 20 seconds, and 68 ° C. for 2 minutes as one cycle.
  • the amplified upstream / downstream ligated fragment of phaP1 Cn was 5′-phosphorylated by kinase treatment, and the vector plasmid pK18mobsacB was cleaved with HincII to prepare pK18ms-P1ud linked to the dephosphorylated fragment.
  • the amplified phaC NSDG fragment was 5′-phosphorylated by kinase treatment and ligated with the vector fragment obtained by removing phaC Ac from pEE32.
  • a plasmid in which phaC NSDG was ligated in the same direction as the upstream and downstream genes was selected and designated pEE32-NSDG.
  • the entire region (7.3 kb) was amplified by PCR using pK18ms-P1ud as a template and the oligonucleotides of sequence 2 and sequence 4 described above as primers, and at the junction of the phaP1 Cn upstream region and phaP1 Cn downstream region.
  • An open vector fragment was obtained.
  • KOD Plus Neo DNA polymerase (Toyobo Co., Ltd.) was used, and the reaction was performed 30 cycles at 98 ° C. for 20 seconds, 63 ° C. for 20 seconds, and 68 ° C. for 4 minutes.
  • the amplified phaP Ac fragment was 5′-phosphorylated by kinase treatment and ligated with a vector fragment opened from pK18ms-P1ud by PCR amplification.
  • a plasmid in which phaP Ac was ligated in the same direction as the promoter region was selected and designated pK18ms-P1ud-P Ac .
  • the amplified phaP Ac -phaC NSDG -phaJ Ac fragment was 5′-phosphorylated by kinase treatment and ligated with the vector fragment opened from the above-mentioned pK18ms-P1ud by PCR amplification.
  • a plasmid in which phaP Ac -phaC NSDG -phaJ Ac was ligated in the same direction as the promoter region was selected and designated pK18ms-P1ud-P Ac CJ.
  • Sequence 12 ATGAGCGCACAATCCCTGGAAGTAG (SEQ ID NO: 16) Next, the vector fragment that had been opened except for phaC NSDG was 5′-phosphorylated by kinase treatment and self-ligated at both ends to prepare pK18ms-P1ud-P Ac J.
  • phaP1 Cn fragment (0.6 kb) (SEQ ID NO: 4) was amplified by PCR using the genomic DNA of necator H16 strain as a template and oligonucleotides of the following sequences 13 and 14 as primers. PCR was performed for 30 cycles, with a reaction of 98 ° C. for 20 seconds, 54 ° C. for 20 seconds, and 68 ° C. for 50 seconds as one cycle.
  • Sequence 13 ATGATCCTCACCCCGGGAACAA (SEQ ID NO: 17)
  • Sequence 14 TCAGGCAGCCCGTCGTCTTCTTTGCCCGT (SEQ ID NO: 18)
  • phaP1 Cn upstream-phaP Ac -phaC NSDG -phaJ Ac -phaP1 Cn downstream fragment (3. p.sup.18 ms-P1ud-P Ac CJ as a template and PCR using the oligonucleotides of sequence 1 and sequence 3 described above as primers. 3 kb) was amplified.
  • the amplified fragment is 5′-phosphorylated by kinase treatment, ligated to the HincII site of pK18mobsacB, a plasmid in which the fragment is ligated in the opposite direction to pK18ms-P1ud-P Ac CJ is selected, and pK18ms-P1ud-P Ac CJ-R.
  • KOD Plus Neo DNA polymerase (Toyobo Co., Ltd.) was used, and the reaction was carried out for 30 cycles, with a reaction of 98 ° C. for 20 seconds, 59 ° C. for 20 seconds, and 68 ° C. for 5 minutes.
  • the amplified phaP1 Cn fragment was 5′-phosphorylated by kinase treatment, and ligated with a vector fragment obtained by removing phaP Ac from K18ms-P1ud-P Ac CJ-R.
  • a plasmid in which phaP1 Cn was linked in the same direction as the promoter region and the downstream gene was selected and designated pK18ms-P1ud-P1 Cn CJ.
  • phaP1 Cn amplified fragment was 5′-phosphorylated by kinase treatment, and ligated with the vector fragment obtained by removing phaP Ac from pK18ms-P1ud-P Ac J.
  • a plasmid in which phaP1 Cn was linked in the same direction as the promoter region and the downstream gene was selected and designated pK18ms-P1ud-P1 Cn J.
  • Example 2 Production and PHA biosynthesis of NSDG-P1 Cn J strain and NSDG-P Ac J strain
  • the gene (phaJ Ac ) encoding R-hydratase derived from Caviae strain is C.I.
  • NSDG-P1 Cn J strain was inserted downstream of phaP1 Cn of NSDG lines from necator strains, further A.
  • This phaP1 Cn is phaC NSDG akin NSDG-P Ac J strain substituted with phaP Ac derived from Caviae strain was prepared, and its synthetic ability was examined. Specifically, it is as follows.
  • C.I. Necator NSDG strain was transformed by conjugation transfer. First, pK18ms-P1ud-P1 Cn J was introduced into Escherichia coli S17-1 by the calcium chloride method. Next, this recombinant Escherichia coli was cultured overnight at 37 ° C. in 3.0 ml of LB medium (1% tryptone, 1% sodium chloride, 0.5% yeast extract, pH 7.2). In parallel with this, C.I. Necator NSDG strain was cultured overnight at 30 ° C.
  • necator NSDG strain 0.1 ml was mixed and cultured at 30 ° C. for 6 hours. This bacterial cell mixture was applied to a Simons Citrate agar medium (Difco) supplemented with 0.2 mg / ml kanamycin and cultured at 30 ° C. for 3 days.
  • the recombinant Escherichia coli plasmid is C.I.
  • the bacterial cells transferred to the necator and incorporated into the chromosome by homologous recombination show kanamycin resistance, while the recombinant Escherichia coli cannot grow on the Simmons Citrate agar medium.
  • PK18ms-P1ud-P1 Cn J incorporated into the chromosome of the recombinant E. coli Necator transformant (pop-in strain). Further, the pop-in strain was cultured in NR medium at 30 ° C. overnight, applied to NR medium supplemented with 10% sucrose, and cultured at 30 ° C. for 3 days.
  • Levansucrase encoded by sacB on the pK18mobsacB-derived vector accumulates toxic polysaccharide in cells using sucrose as a substrate. For this reason, only a strain from which the plasmid region has been eliminated (pop-out strain) can grow in a medium supplemented with 10% sucrose. From these colonies, a clone in which phaJ Ac was inserted downstream of phaP1 Cn on the chromosome was selected by the PCR method, and this was designated NSDG-P1 Cn J strain.
  • NSDG strain was similarly transformed by conjugation transfer. PhaP1 Cn on the chromosome from the colonies resulting pop-out line was replaced in phaP Ac, further Clones PhaJ Ac is inserted into the downstream were selected by PCR, which the NSDG-P Ac J strain did.
  • Recombinant strains pre-cultured in NR medium were treated with 100 ml of MB medium (0.9% disodium hydrogen phosphate 12 hydrate, 0.15% potassium dihydrogen phosphate, 0.05% ammonium chloride, % Trace metal solution) and cultured with shaking in a Sakaguchi flask at 30 ° C. for 72 hours. 1% soybean oil was used as a carbon source. In addition, 0.1 mg / ml kanamycin is added to the medium. After culturing, the cells were collected by centrifugation, washed with 70% ethanol to remove the attached oil, and then washed with distilled water. The obtained cells were freeze-dried and the dry cell weight was measured.
  • the temperature was raised from the initial temperature of 100 ° C. at a rate of 8 ° C./min.
  • the prepared recombinant C.I. Table 1 shows the composition of related genes on the chromosome of the necator strain and the results regarding PHA production.
  • phaC NSDG was introduced into pha operon on the chromosome, further NSDG-P1 Cn J strain was inserted PhaJ Ac to phaP1 Cn downstream on the chromosome, soy oil carbon source 3HHx fraction of 6.5 mol% P ( 3HB-co-3HHx) was synthesized.
  • PHA synthase is A.I. derived from the Caviae strain and the major granule-binding protein is C.I. It is different from the necator strain.
  • phaP1 Cn on the chromosome is converted to the same A.P.
  • NSDG-P Ac J strain which was phaP Ac derived from Caviae strain, the 3HHx fraction increased from 6.5 mol% to 9.3 mol% while the PHA production amount increased slightly.
  • Example 3 Production of NSDG-P1 Cn CJ strain and NSDG-P Ac CJ strain and biosynthesis of PHA
  • NSDG-P1 Cn J strain phaP1 Cn and phaJ Ac were examined for their PHA biosynthesis ability. Specifically, it is as follows.
  • NSDG strain was similarly transformed by conjugation transfer.
  • a clone in which phaC NSDG -phaJ Ac was inserted downstream of phaP1 Cn on the chromosome was selected from the obtained popout colonies by PCR, and this was designated NSDG-P1ud-P1 Cn CJ strain.
  • NSDG strain was similarly transformed by conjugative transfer using the recombinant plasmid pK18ms-P1ud-P Ac CJ obtained in Example 1 (4).
  • PhaP1 Cn on the chromosome from the colonies resulting pop-out line was replaced in phaP Ac, further Clones phaC NSDG -phaJ Ac is inserted into the downstream were selected by PCR, which NSDG-P Ac CJ strain was designated.
  • Table 2 shows the composition of related genes on the chromosome of the prepared recombinant strain and the results relating to PHA production.
  • the phaC NSDG introduced into pha operon on the chromosome, NSDG-P1 Cn CJ strains inserting the phaC NSDG and PhaJ Ac further 1 copy phaP1 Cn downstream on the chromosome, 3HHx fraction from soybean carbon source 10 .5 mol% P (3HB-co-3HHx) was synthesized.
  • PHA synthase is A.I. derived from the Caviae strain and the major granule-binding protein is C.I. It is different from the necator strain.
  • phaP1 Cn on the chromosome is converted to the same A.P.
  • NSDG-P Ac CJ strain which is phaP Ac derived from Caviae strain
  • the 3HHx fraction increased from 10.5 mol% to 17.2 mol% while the PHA production amount increased slightly.
  • Example 4 Production of MF02-P Ac strain and PHA biosynthesis
  • the previously produced MF02 strain is a strain in which phaC NSDG -phaJ Ac is introduced into the chromosome pha operon, and 4.6 mol% 3HHx from soybean oil.
  • a copolymer is biosynthesized (Patent Document 6).
  • a MF02-P Ac strain in which the main granule-binding protein gene phaP1 Cn was replaced with phaP Ac was prepared, and its PHA biosynthesis ability was examined. Specifically, it is as follows.
  • the MF02 strain was similarly transformed by conjugation transfer.
  • a clone in which phaP1 Cn on the chromosome was replaced by phaP Ac was selected from the obtained popout colonies by PCR, and this was designated as MF02-P Ac strain.
  • the MF02 strain which is derived from different PHA synthase and main granule binding protein, synthesized P (3HB-co-3HHx) having a 3HHx fraction of 4.6 mol% from a soybean oil carbon source.
  • phaP1 Cn on the chromosome of MF02 strain with phaP Ac derived from the same as phaC NSDG , the origin of the PHA synthase and the main granule binding protein is the same
  • the 3HHx fraction increased from 4.6 mol% to 6.4 mol% while the PHA production amount increased slightly.
  • the strain with the same microbial species from both has improved the PHA production amount, and the 3HHx fraction is 1.4 to 1.7 times could be increased.
  • the NSDG-P Ac J strain can biosynthesize about 9 mol% 3HHx-containing copolymer PHA that can be expected to show an appropriate degree of crystallinity and flexibility, compared to conventional strains.
  • NSDG-P Ac CJ strain efficiently biosynthesizes P (3HB-co-3HHx) with a high 3HHx fraction of 17.2 mol%.
  • Such a copolymerized PHA having a high 3HHx fraction has a potential for new applications as a highly flexible bioplastic.
  • an excellent copolymerized polyhydroxyalkanoic acid producing strain and a method for efficiently producing a copolymerized polyhydroxyalkanoic acid having a high 3HHx fraction are provided.
  • a method for controlling the composition ratio of the monomer units can be provided.

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Abstract

Le but de la présente invention est de fournir un procédé qui permet la régulation de la composition relative en unités monomères dans un copolymère d'acide polyhydroxyalcanoïque. L'invention concerne un procédé qui permet la régulation de la composition relative en unités monomère d'un copolymère PHA, et dans lequel, au sein d'une souche recombinante rendue productrice du copolymère de PHA par insertion d'un ou plusieurs gènes qui codent pour une enzyme contrôlant la production de polymère d'acide polyhydroxyalcanoïque (PHA) à spécificité de substrat large dans un hôte différent de l'organisme vivant du gène inséré, un gène qui code pour la principale protéine de liaison aux granules de PHA de la souche recombinante est transformé en un gène qui code pour une protéine de liaison aux granules de PHA dérivée du même organisme vivant que celui du gène qui code pour l'enzyme du polymère de PHA.
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CN114381415B (zh) 2022-03-22 2022-11-15 深圳蓝晶生物科技有限公司 高产pha的基因重组菌及其构建方法

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