WO2016021604A1 - 糖質原料からの共重合ポリヒドロキシアルカン酸の製造法 - Google Patents
糖質原料からの共重合ポリヒドロキシアルカン酸の製造法 Download PDFInfo
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Definitions
- the present invention relates to the production of poly (3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid), which is one of copolymerized polyhydroxyalkanoic acids that are degradable by microorganisms and excellent in biocompatibility, by microorganisms.
- the present invention relates to a method for improving the production amount of a copolymer and improving the fraction of 3-hydroxyhexanoic acid in the copolymer using sugar and / or glycerol as a basic raw material.
- Plastic is an indispensable material in modern society because it can realize various physical properties depending on the structure and is inexpensive.
- most plastics are synthesized using petroleum as a raw material, and have been developed and produced for long-term stability.
- many petroleum synthetic plastics are not decomposed in the natural environment after disposal, and management and disposal of plastic waste that has become unnecessary has become a major problem in countries around the world.
- the depletion of fossil resources represented by oil is an exploratory problem. Even if fossil resources are available for years due to advances in mining technology, they remain limited resources, and consumption of fossil resources will continue to expand as a result of growing demand resulting from global economic growth. It is predicted to go.
- the increase in atmospheric carbon dioxide concentration due to the consumption of fossil resources is also a major environmental problem. Therefore, it is necessary to control the consumption of fossil resources and to build a social system that does not depend on fossil resources.
- Bioplastic is a general term for two types of biodegradable plastic and biomass plastic.
- Biodegradable plastics are plastics that are decomposed by microorganisms in the environment, and do not cause waste disposal problems that are a problem with petroleum synthetic plastics.
- Biomass plastic is a plastic made from renewable biomass resources derived from animals and plants, and the carbon dioxide produced by combustion is carbon dioxide in the atmosphere that was originally immobilized by plant photosynthesis (carbon neutral). In terms of fossil resource depletion and greenhouse gas emissions, this material can contribute to the construction of a future recycling society.
- Polyhydroxyalkanoic acid in which many microorganisms accumulate in cells as an energy source, is expected to be a plastic material that uses biomass as a raw material and is biodegradable.
- Poly (3-hydroxybutanoic acid) [P (3HB)] is a typical PHA biosynthesized by many microorganisms, but P (3HB) is hard and brittle, making it difficult to put it into practical use as a material.
- PHA copolymers having hydroxyalkanoic acids having different structures as comonomer units have improved flexibility, research on biosynthesis of PHA copolymers has been vigorously conducted so far. .
- a poly (3-hydroxybutanoic acid-co-3-hydroxyvaleric acid) copolymer is converted into 1,4-butanediol or ⁇
- a poly (3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) copolymer is biosynthesized by adding butyllactone.
- Poly ((R) -3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) copolymer [P (), which is biosynthesized by cultivating soil bacteria such as Aeromonas caviae (Aeromonas caviae) using vegetable oil or fatty acid as a carbon source 3HB-co-3HHx)] increases in flexibility depending on its copolymer composition, and a copolymer containing about 10 mol% of (R) -3-hydroxyhexanoic acid (3HHx) units exhibits moderate flexibility. It turns out to be an excellent plastic.
- P () which is biosynthesized by cultivating soil bacteria such as Aeromonas caviae (Aeromonas caviae) using vegetable oil or fatty acid as a carbon source 3HB-co-3HHx)
- increases in flexibility depending on its copolymer composition and a copolymer containing about 10 mol% of (R) -3-hydroxyhexanoic acid (3HHx) units exhibits moderate
- 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).
- the hydrogen bacterium Cupriavidus necator known as an efficient P (3HB) producing fungus functions as a substrate-specific PHA-polymerizing enzyme, and ⁇ -oxidation that degrades the polyester biosynthetic pathway and fatty acids.
- Recombinant strains have been developed that efficiently produce P (3HB-co-3HHx) with a high 3HHx fraction using vegetable oil as a carbon source by modifying the pathway (Patent Document 3, Patent Document 4, Patent Document 5) , Patent Literature 6, Non Patent Literature 2, Non Patent Literature 3, Non Patent Literature 4, and Non Patent Literature 5).
- crotonyl-CoA is produced from (R) -3HB-CoA.
- a gene phaJ Ac of (R) -specific enoyl-CoA hydratase derived from Caviae and a gene ccr Sc of crotonyl-CoA reductase derived from Streptomyces cinnamonensis (Streptomyces cinnamonensis) that reduces the double bond of crotonyl-CoA Along with the gene phaC Ac of the broad substrate-specific PHA synthase, C.I. This was introduced into a nematic PHA synthase-deficient strain PHB - 4.
- the prepared recombinant strain was designed by biosynthesizing P (3HB-co-3HHx) containing 1.2 to 1.6 mol% of 3HHx units with fructose as a single carbon source at 39 to 49% by weight.
- the route was found to work, but the 3HHx fraction was low and not sufficient to improve polymer properties (Non-Patent Document 8).
- JP-A-5-93049 Japanese Patent Laid-Open No. 7-265065 Japanese Patent No. 3062459 JP 2008-86238 A JP 2008-29218 A International Publication No. 2011/105379
- the acetoacetyl-CoA reductase gene (phaB1) present in the phaCAB1 operon of the necator strain was deleted, the crotonyl-CoA reductase gene was incorporated, and the (R) -specific enoyl-CoA hydratase gene and ethylmalonyl-CoA decarboxylation Recombinant C into which an enzyme gene has been incorporated
- the necator strain was found to be able to produce P (3HB-co-3HHx) having a high 3HHx fraction with a high accumulation rate using carbohydrates and glycerol as raw materials, and the present invention was completed.
- the present invention is as follows.
- Transforming the genetic enoyl-CoA hydratase gene and the ethylmalonyl-CoA decarboxylase gene by homologous recombination, or by introducing an autonomously replicating vector incorporating the gene into the strain
- a method for producing poly (3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) comprising growing a transformant in a medium containing saccharide and / or glycerol as [2] Recombinant C.
- a chromosome of a recombinant strain lacking a gene encoding acetoacetyl-CoA reductase on the chromosome of a necator strain is transformed by crotonyl-CoA reductase gene by homologous recombination, or the gene is transformed into the gene
- Poly (3-hydroxybutanoic acid-co- which comprises transforming by introducing an autonomously replicating vector into which is incorporated and growing the transformant in a medium containing carbohydrate and / or glycerol as a carbon source. 3-hydroxyhexanoic acid).
- the method further comprises transforming the (R) -specific enoyl-CoA hydratase gene by homologous recombination or introducing an autonomously replicating vector into which the gene is incorporated.
- the method further comprises transforming the ethylmalonyl-CoA decarboxylase gene by homologous recombination, or by introducing an autonomously replicating vector into which the gene has been incorporated. [2] and [3].
- the crotonyl-CoA reductase gene is (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 1; or (b) hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions and from crotonyl-CoA to butyryl-
- the crotonyl-CoA reductase gene is (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 2; or (b) hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 2 under stringent conditions and from crotonyl-CoA to butyryl-
- the (R) -specific enoyl-CoA hydratase gene is C.I.
- the ethylmalonyl-CoA decarboxylase gene is (A) a nucleic acid comprising the base sequence represented by SEQ ID NO: 4; or (b) hybridizing with a nucleic acid comprising the base sequence represented by SEQ ID NO: 4 under stringent conditions and removing ethylmalonyl-CoA.
- C The schematic diagram of the genotypes of necator H16 strain (wild strain), MF01 strain, MF01 ⁇ B1 strain, MF01 ⁇ B1B3 strain is shown.
- C.I. introduced pBPP-ccrMeJ4a-emd The P (3HH-co-3HHx) biosynthesis pathway from fructose in necator MF01 ⁇ B1 is shown.
- the present inventors broadly started carbohydrates and glycerol, unlike conventional vegetable oils, by developing 3HB units and 3HHx units from acetyl-CoA and by developing new metabolic pathways for copolymerization.
- the present invention relates to recombinant C.I. to which P (3HH-co-3HHx) production ability is imparted.
- a chromosome of a necator strain is transformed by homologous recombination with a crotonyl-CoA reductase gene, a (R) -specific enoyl-CoA hydratase gene, and an ethylmalonyl-CoA decarboxylase gene;
- a method for producing P (3HH-co-3HHx) comprising transforming by introducing an autonomously replicating vector into which is incorporated, and growing the transformant in a medium containing carbohydrate or glycerol as a carbon source And a method for improving the production amount of the copolymer and / or the fraction of 3HHx units in the copolymer.
- the crotonyl-CoA reductase gene, the (R) -specific enoyl-CoA hydratase gene, and the ethylmalonyl-CoA decarboxylase gene are all transformed by homologous recombination, or
- the strain may be introduced as an autonomously replicating vector in which the gene is incorporated, or the crotonyl-CoA reductase gene, the (R) -specific enoyl-CoA hydratase gene, and the ethylmalonyl-CoA decarboxylase gene.
- one or two genes may be transformed by homologous recombination, and the remaining genes may be introduced as autonomously replicating vectors in which the genes are incorporated into the strain.
- C.I. The gene encoding acetoacetyl-CoA reductase on the chromosome of the necator strain may be deleted.
- the present invention relates to recombinant C.I. to which P (3HH-co-3HHx) production ability is imparted.
- a chromosome of a recombinant strain lacking a gene encoding acetoacetyl-CoA reductase on the chromosome of a necator strain is transformed by crotonyl-CoA reductase gene by homologous recombination, or the gene is transformed into the gene
- a method for producing P (3HH-co-3HHx) comprising transforming by introducing an autonomously replicating vector into which is incorporated, and growing the transformant in a medium containing carbohydrate or glycerol as a carbon source And a method for improving the production amount of the copolymer and / or the fraction of 3HHx units in the copolymer.
- the transformant used in the production method described above is transformed by homologous recombination of the (R) -specific enoyl-CoA hydratase gene, or by introducing an autonomously replicating vector incorporating the gene. It may be transformed. Furthermore, the transformant used in the above production method is obtained by homologous recombination of the ethylmalonyl-CoA decarboxylase gene, or the (R) -specific enoyl-CoA hydratase gene and the ethylmalonyl-CoA decarboxylase gene. It may be transformed or transformed by introducing an autonomously replicating vector incorporating the gene.
- the host used in the production method of the present invention is C. a. necator.
- C.I. used in the production method of the present invention.
- Necators include, but are not limited to, JMP134 strain (DSM4058) and H16 strain (DSM428). More specifically, in the present invention, recombinant C.I. to which P (3HB-co-3HHx) producing ability is imparted.
- Necator strains are preferably used. For example, NSDG strain, MF01 strain, and NSDG ⁇ A strain may be used.
- NSDG strain refers to C.I.
- phaC NSDG means A.I.
- MF01 strain is a transformant in which phaA Cn of the NSDG strain is replaced with a broad substrate-specific ⁇ -ketothiolase gene bktB Cn .
- NSDG ⁇ A refers to a transformant in which the phaA Cn that is a ⁇ -ketothiolase gene is deleted from the NSDG strain.
- the three H16 mutants are C.I. Based on the sequence information of the gene encoding necata PHA synthase, it can be prepared using a general genetic engineering technique (see, for example, JP2008-29218, International Publication WO2011 / 105379) ).
- crotonyl-CoA reductase (ccr)
- the recombinant C.I. It is necessary to introduce a gene (ccr) encoding crotonyl-CoA reductase into a necator strain by transformation.
- crotonyl-CoA reductase refers to the reduction of crotonyl-CoA having 4 carbon atoms, which is an intermediate in the fatty acid ⁇ -oxidation pathway, and a substrate of ⁇ -ketothiolase (BktB).
- butylyl-CoA is condensed with another molecule of acetyl-CoA by the action of ⁇ -ketothiolase, and further converted to supply (R) -3HHx-CoA having 6 carbon atoms, and polyester polymerization showing a wide substrate specificity Copolymerized with (R) -3HB-CoA by the enzyme.
- the origin of the biological species of ccr that can be used in the present invention is not particularly limited as long as the reductase after translation has the above-mentioned activity.
- ccr Sc a gene encoding crotonyl-CoA reductase derived from cinnamonensis
- ccr Me A gene encoding crotonyl-CoA reductase derived from extorquens
- the ccr 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.
- any base sequence of a nucleic acid encoding crotonyl-CoA reductase can be used. Nucleic acids having sequences are also within the scope of the present invention.
- the base sequence of ccr Sc GenBank Accession No. AF178673 and ccr Me base sequence: NCBI-GeneID: 7990208 can be used. Isolation and identification of ccr can be carried out by ordinary molecular biological techniques. These genes can be amplified using genomic DNA as a template by designing synthetic nucleotides as primers based on the nucleotide sequence of SEQ ID NO: 1 or 2, as described in Example 1 described later.
- ccr Sc when the primers of SEQ ID NOs: 11 and 12 are used, a DNA fragment of about 1.3 kbp is obtained as a PCR product.
- these are agarose gel electrophoresis.
- Nucleic acids can be isolated according to a conventional method such as a method of separating DNA fragments by a molecular weight such as a method of cutting out a specific band.
- the ccr Me using primers of SEQ ID NO: 13 and 14, can be isolated as well.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand displacement reactions
- ccr is S.I. cinnamonensis derived from (a) a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 1; or (b) hybridizing with a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions; and It may be composed of a nucleic acid encoding a protein having a catalytic activity to produce butyryl-CoA from crotonyl-CoA.
- ccr is M.I.
- nucleic acid containing a nucleotide sequence represented by SEQ ID NO: 2 or (b) hybridizing with a nucleic acid comprising a nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions; It may be composed of a nucleic acid encoding a protein having a catalytic activity to produce butyryl-CoA from crotonyl-CoA.
- 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, with respect to the base sequence described in SEQ ID NO: 1 or 2. 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. Alternatively, the percent identity of two nucleic acid sequences is determined by Devereux, J. et al. , 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.
- ccr introduced into the host may be ccr Sc or ccr Me , and may be a nucleic acid comprising the base sequence represented by SEQ ID NO: 1 or 2.
- 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.
- a promoter of a different species from the host may be introduced into the chromosome by genetic engineering and used as appropriate.
- a gene (phaJ) encoding (R) -specific enoyl-CoA hydratase is further introduced into a host into which the gene encoding crotonyl-CoA reductase is introduced. Also good.
- “(R) -specific enoyl-CoA hydratase” used in the present invention is a fatty acid ⁇ -oxidation intermediate, 2-enoyl-CoA, which is a PHA monomer (R) -3-hydroxy. It means an enzyme that converts to acyl-CoA, and the origin of the biological species is not particularly limited as long as it has this activity. Necator strain.
- 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 multiple base sequences encoding one amino acid.
- the base sequence of a nucleic acid encoding (R) -specific enoyl-CoA hydratase may be used.
- a nucleic acid having any base sequence is included in the scope of the present invention.
- examples of phaJ used in the present invention include C.I. H16 from necator A1070 (hereinafter referred to as “phaJ4a”) (NCBI-GeneID: 4248869) can be used.
- phaJ4a C.I. H16 from necator A1070
- isolation and identification of phaJ introduced into a host can be performed by a normal molecular biological technique.
- 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 under stringent conditions.
- 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. It may be composed of a nucleic acid that encodes a protein that hybridizes under conditions and has an activity of converting a fatty acid ⁇ -oxidation intermediate into (R) -3-hydroxyacyl-CoA.
- 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 gene encoding (R) -specific enoyl-CoA hydratase introduced into the host is phaJ4a, and may be a nucleic acid comprising the base sequence represented by SEQ ID NO: 3. Good.
- 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 promoter instead of the promoter inherent in the host chromosome, a promoter of a different species from the host may be introduced into the chromosome by genetic engineering and used as appropriate.
- Ethylmalonyl-CoA decarboxylase gene (emd)
- the above-described crotonyl-CoA reductase encoding gene is introduced into a host, instead of the gene encoding (R) -specific enoyl-CoA hydratase (phaJ) or
- an ethylmalonyl-CoA decarboxylase gene (emd) may be introduced.
- ethylmalonyl-CoA decarboxylase used in the present invention is a catalyst for decarboxylation of ethylmalonyl-CoA to butyryl-CoA produced by side reaction with propionyl-CoA carboxylase or the like in animal cells.
- the origin of the biological species is not particularly limited.
- the emd 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, but any nucleic acid base sequence encoding ethylmalonyl-CoA decarboxylase may be used. Nucleic acids having the base sequences are also included in the scope of the present invention.
- the emd used in the present invention is, for example, an amino acid sequence of mouse-derived ethylmalonyl-CoA decarboxylase (GenBank Accession No. NP). 001103665), an artificially back-translated gene (hereinafter referred to as “emd Mm ”) can be used.
- emd Mm an artificially back-translated gene
- Such artificial genes can be obtained by gene synthesis by various companies.
- emd 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 under stringent conditions.
- emd 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 and stringent. It may consist of a nucleic acid that hybridizes under conditions and encodes a protein having catalytic activity to decarboxylate ethynylmalonyl-CoA to produce butyryl-CoA.
- the “stringent conditions” are as described above, and the homologous nucleic acid cloned using a nucleic acid amplification reaction, hybridization, or the like is the base sequence described in SEQ ID NO: 4, respectively. 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 ethylmalonyl-CoA decarboxylase gene introduced into the host may be an emd Mm nucleic acid comprising the base sequence represented by SEQ ID NO: 4.
- 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 promoter instead of the promoter inherent in the host chromosome, a promoter of a different species from the host may be introduced into the chromosome by genetic engineering and used as appropriate.
- acetoacetyl-CoA reductase a recombinant C. elegans gene lacking a gene encoding acetoacetyl-CoA reductase has been deleted. It is preferable to use necator strains.
- the “acetoacetyl-CoA reductase” is an enzyme having a catalytic function to produce (R) -3HB-CoA using acetoacetyl-CoA as a substrate.
- PhaB1 In necator, “PhaB1”, “PhaB2”, and “PhaB3” are known, and in the above reaction under the growth conditions in fructose, PhaB1 mainly functions, but PhaB3 that is a paralog also functions. Have been reported (Budde, C.F., et al., J. Bacteriol., 192: 5319-5328 (2010)). As described above, the recombinant C.I. used in the production method of one embodiment of the present invention.
- a strain lacking a gene encoding acetoacetyl-CoA reductase is used as a necator strain, and the aceacetyl-CoA reductase to be deleted is preferably encoded by phaB1 or phaB1 and phaB3. Yes, more preferably phaB1.
- the nucleotide sequences of the genes to be deleted are known. For example, by referring to NCBI-GeneID: 4249784 and NCBI-GeneID: 4250155, respectively, either or both of these genes can be identified. Deleted recombinant C.I. It can be used to obtain necator strains.
- deletion means a state in which a part or all of a gene of interest has disappeared due to genetic manipulation, and as a result, the protein encoded by the gene. It is intended that some or all of the activity is lost.
- the deletion mutation can be performed by a known site mutagenesis method (Current Protocols in Molecular Biology 1, Volume 8.1.1, 1994) or a commercially available kit (LA PCR in vitro Mutagenesis series of Takara). Kit).
- each or any combination of these genes for introducing ccr, phaJ, and emd into the host chromosome is homologous.
- a gene replacement vector incorporated into a replacement vector or an expression vector incorporating each or any combination of the genes into an autonomously replicating vector is provided.
- a method for incorporating a gene into a vector for example, Sambrook, J. et al. Et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 1.1 (2001).
- a commercially available ligation kit for example, manufactured by Toyobo Co., Ltd. can also be used.
- 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.
- the vector for homologous recombination pK18mobsacB (Schaffer, A., et al., Gene, 145: 69-73 (1994)), pJQ200 (Quantt, J. and Hynes) , MP, “Versatile suclide vectors whoh select direct selection for gene replacement in gram-negative bacteria”, Gene (1 93) 127: 15-21
- pBBR1-MCS2 GeneBank Accession No. U23751
- pJRD215 M16198
- E. coli include, for example, pBAD24 (GenBank Accession No. X81837), pDONR201, pBluescript, p It is possible to use the C118, pUC18, pUC19, pBR322 and the like.
- 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 that homologous recombination occurs with the gene of the target host cell (if necessary, an autonomous replication origin, a conjugative transfer region, a selectable 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.
- a conjugation transfer method can be used to introduce an expression vector 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.
- a crotonyl-CoA reductase gene is introduced into the chromosome of a recombinant strain in which the gene encoding acetoacetyl-CoA reductase of necator strain has been deleted, and (R) -specific enoyl-CoA hydratase gene or ethylmalonyl -By introducing a CoA decarboxylase gene, the copolymer is produced and accumulated in the recombinant strain or in a culture (for example, a medium), and the desired co-polymer is obtained from the recombinant strain or culture. This is done by collecting the coalescence.
- the recombinant strain in order to synthesize the copolymer, it is preferable to place the recombinant strain under appropriate culture conditions.
- 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 carbohydrate and / or glycerol as a carbon source.
- the medium when the necator strain is used as a host include a medium in which a saccharide or glycerol that can be assimilated by the microorganism strain is added, and any of nitrogen sources, inorganic salts, and other organic nutrient sources is restricted.
- the medium temperature is in the range of 25 ° C. to 37 ° C., and aerobically cultured for 1 to 10 days, so that the copolymer is produced and accumulated in the cells, and then recovered and purified. A desired copolymer can be produced.
- sugar when using saccharide
- the supply source is not specifically limited.
- “Sugar” is a polyhydric alcohol having an aldehyde group or a ketone group, and means a monosaccharide, oligosaccharide, oligosaccharide, or sugar derivative. Specific examples of monosaccharides include glucose, galactose, mannose, glucosamine, N-acetylglucosamine, and fructose.
- disaccharide examples include maltose, isomaltose, lactose, lactosamine, N-acetyllactosamine, cellobiose, melibiose and the like.
- Oligosaccharides include homo-oligomers composed of glucose, galactose, mannose, glucosamine, N-acetylglucosamine, fructose, etc., or two components such as glucose, galactose, mannose, glucosamine, N-acetylglucosamine, fructose, sialic acid
- Hetero-oligomers composed of the above are mentioned, and examples thereof include maltooligosaccharides, isomaltooligosaccharides, lactoligosaccharides, lactosamine oligosaccharides, N-acetyllactosamine oligosaccharides, cellooligosaccharides, and merbiooligosaccharides.
- polysaccharides include those found in a wide range of organisms such as animals, plants (including seaweed), insects, and microorganisms.
- N-linked sugar chains O-linked sugar chains, glycosaminoglycans, starches Amylose, amylopectin, cellulose, chitin, glycogen, agarose, alginic acid, hyaluronic acid, inulin, glucomannan and the like.
- sugar derivatives include deoxyribose (C 5 H 10 O 4 ) and sulfated polysaccharides.
- the sugar concentration in the medium is preferably 0.1 to 5%, but can be appropriately adjusted by those skilled in the art.
- Glycerol is often used interchangeably with “glycerin”. More suitably, however, “glycerol” is applied to 1,2,3-propanetriol, which is a chemically pure compound, whereas “glycerin” has been purified with a glycerol content generally greater than 95%. Applicable to commercial products. According to the present invention, it may be any when used as a carbon source. The concentration of glycerol or glycerin in the medium is preferably 0.1 to 5%, but can be appropriately adjusted by those skilled in the art. In addition, the present invention does not exclude an embodiment in which a carbohydrate and glycerol (or glycerin) are mixed and used as a carbon source.
- 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.
- the copolymer can be purified as follows: The transformant is recovered from the medium by centrifugation and washed with distilled water. Thereafter, it is dried or freeze-dried. Thereafter, the transformant dried in chloroform is suspended, and stirred at room temperature for a predetermined time to extract the copolymer. In the extraction stage, heating may be performed if necessary. The residue is removed by filtration, methanol is added to the supernatant to precipitate the copolymer, and the precipitate is filtered or centrifuged to remove the supernatant and dried to obtain a purified copolymer. Then, although not limited, the composition ratio of the monomer units of the obtained copolymer can be confirmed using NMR (nuclear magnetic resonance) and gas chromatography.
- the 3HHx fraction in P (3HB-co-3HHx) may be at least 1% mol or more, for example, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol% It may be 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 copolymer obtained by the control method of the present invention is accumulated in the cells at a rate of 20 to 95% by weight, preferably 40 to 95% by weight, based on the dry cell weight.
- Example 1 C. as a host Necator strain and crotonyl-CoA reductase (1) Host The necator wild-type strain H16 has a phaCAB1 operon encoding PHA synthase (PhaC), ⁇ -ketothiolase (PhaA), and acetoacetyl-CoA reductase (PhaB1) constituting the P (3HB) biosynthesis pathway on the chromosome. Have. C. produced previously. necator MF01 strain is a phaC in the phaCAB1 operon of H16 strain. phaA was converted to C. aeruginosa to the mutated gene phaC NSDG of PHA synthase derived from Caviae.
- PhaC PHA synthase
- PhaA ⁇ -ketothiolase
- PhaB1 acetoacetyl-CoA reductase
- PCR basically uses KOD Plus DNA polymerase (manufactured by Toyobo Co., Ltd.). One cycle of reaction at 98 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 68 ° C. for 2 minutes and 30 seconds. This was performed for 30 cycles, and the temperature conditions were adjusted as necessary.
- plasmid vector for deletion of acetoacetyl-CoA reductase gene phaB1 The plasmid pK18msbktBR for homologous recombination for deleting the gene (phaB1) encoding acetoacetyl-CoA reductase PhaB1 on the chromosome of necator MF01 strain was prepared as follows. First, C.I. by PCR using the genomic DNA fragment of Necator H16 strain as a template and oligonucleotides of the following sequences 1 and 2 as primers. Necator-derived ⁇ -ketothiolase (BktB) was amplified.
- Sequence 1 TACATGGATCCAAGGGAGGCAAAGTCATGACGCGGTGAAGTGGGTAGTG (SEQ ID NO: 5)
- Sequence 2 GGATCATATGCTTTCCTCAGATACGCTCGAGATATGGC (SEQ ID NO: 6)
- pK18msNSDG-R including a DNA fragment in which phaC NSDG and phaB1 downstream region were linked
- Plasmid pK18ms ⁇ phaB3 for homologous recombination for deleting the gene (phaB3) encoding acetoacetyl-CoA reductase PhaB3 on the chromosome of necator MF01 strain was prepared as follows. First, C.I. by PCR using the genomic DNA fragment of Necator H16 strain as a template and oligonucleotides of the following Sequence 3 and Sequence 4, and Sequence 5 and Sequence 6 as primers. The upstream region and the downstream region of phaB3 derived from the necator strain were each amplified.
- Sequence 3 TTTGGAATTCTACCTAGGGATCAAATTAGAGGAAA (SEQ ID NO: 7)
- Sequence 4 CCTTACTGCATGTGCCTGCTTCATTCTCGTAAAGTTGAAAG (SEQ ID NO: 8)
- Sequence 5 GAATGAAGCAGGCACATGCAGTAAGGGTGCTGGGG (SEQ ID NO: 9)
- Sequence 6 TCCTAAGCTTGCTGACCGTGATCCGTCGACAACTTTGAAGACCCTGA (SEQ ID NO: 10)
- regions 4 and 5 are overlapped with each other.
- the upstream and downstream fragments of the amplified phaB3 were purified and mixed, and the fragment in which the upstream and downstream of phaB3 were linked by the fusion PCR method using the oligonucleotides of sequence 3 and sequence 6 as primers.
- the amplified upstream / downstream ligated fragment of phaB3 was treated with EcoRI and HindIII. This restriction enzyme-treated fragment was ligated with a vector plasmid pK18mobsacB treated with EcoRI and HindIII to obtain a phaB3 deletion plasmid pK18ms ⁇ phaB3.
- C.I. Necator MF01 strain was cultured overnight at 30 ° C. in 3.0 ml of NR medium (1% fish meat extract, 1% polypeptone, 0.2% yeast extract). Thereafter, C.I. A 0.1 ml culture of necator MF01 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.
- 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 strain in which homologous recombination at the target site occurred on the chromosome was selected by the PCR method.
- the MF01 ⁇ B1 strain lacking phaB1 was transformed by transforming the MF01 strain using pK18msbktBR, and the MF01 ⁇ B3 strain lacking phaB3 was obtained by transforming the MF01 strain using pK18ms ⁇ phaB3. Furthermore, MF01 ⁇ B1B3 strain in which phaB1 and phaB3 were double deleted was obtained by transforming MF01 ⁇ B1 strain with pK18ms ⁇ phaB3 (FIG. 1).
- crotonyl-CoA reductase gene ccr expression vector
- An expression vector pBBR-ccrSc arranged downstream of the lac promoter of plasmid pBBR1-MCS2 capable of autonomous replication in necator cells was prepared.
- methanol-utilizing bacteria M methanol-utilizing bacteria
- extorquens-derived crotonyl-CoA reductase gene (ccr Me )
- An expression vector arranged downstream of the lac promoter of the plasmid pBBR1-MCS2 capable of autonomous replication in necator cells, pBBR-ccrMe, and an expression vector abolished downstream of the phaP1 promoter of pBPP, pBPP-ccrMe were prepared. More specifically, it is as follows.
- Sequence 7 ACGAATTCAGGAGGAACCTGGATGAAGGAAATCCTGACG (SEQ ID NO: 11)
- Sequence 8 AGGTCTAGAGTGCGTTCAGACGTGGCGA (SEQ ID NO: 12)
- the amplified ccr Sc fragment was treated with restriction enzymes EcoRI and XbaI.
- This restriction enzyme-treated fragment was ligated with a fragment obtained by treating pBBR1-MCS2 with restriction enzymes EcoRI and XbaI to obtain a plasmid pBBR-ccrSc for ccr Sc expression.
- the restriction enzyme-treated fragment was ligated with a fragment obtained by treating pBBR1-MCS2 with restriction enzymes EcoRI and XbaI to obtain a plasmid pBBR-ccrMe for expression of ccr Me .
- This restriction enzyme-treated fragment was ligated with a fragment obtained by treating pBPP-ccrMe with the restriction enzyme HindIII to obtain a plasmid pBPP-ccrMeJ4a (FIG. 2) for coexpression of ccr Me and phaJ4a.
- Necator MF01 strain is considered to have a very strong pathway for supplying (R) -3HB-CoA, which is a C4 unit, and MF01 ⁇ B1 strain (FIG. 1) in which phaB1 is further deleted from the modified pha operon of MF01 strain was prepared.
- both ccr Sc and ccr Me were effective in incorporating 3HHx units into PHA, but the ccr Me- introduced strain had a higher 3HHx fraction and a PHA accumulation rate than the ccr Sc- introduced strain. And production was low.
- pBPP contains the lac promoter region of pBBR1-MCS2 in C.I. This is an expression plasmid substituted with the necator-derived phaP1 promoter (Fukui, T., et al., Appl. Microbiol. Biotechnol., 89: 1527-1536 (2011)).
- An expression vector pBPP-ccrMe (FIG. 2) in which ccr Me was inserted into pBPP was prepared. Necator MF01 strain, MF01 ⁇ B1 strain, and MF01 ⁇ B1B3 strain were each transformed.
- Example 2 Improvement of biosynthesis pathway of P (3HB-co-3HHx) from carbohydrate raw material As described above, the expression of exogenous ccr is accompanied by a decrease in PHA production, so P (3HB-co-3HHx) biosynthesis The route was improved.
- C.I For the purpose of strengthening the supply of both (R) -3HB-CoA and (R) -3HHx-CoA, C.I.
- PhaJ4a generates (R) -3HB-CoA and (R) -3HHx-CoA by catalyzing an (R) -specific hydration reaction on enoyl-CoA of C4 and C6.
- It has been reported that it exhibits higher activity against C6 enoyl-CoA than C4 International Publication WO2011 / 105379; Kawashima, Y., et al., Appl. Environ. Microbiol., 78: 493-). 502 (2012)).
- An expression vector pBPP-ccrMeJ4a (FIG. 2) in which phaJ4a was inserted into pBPP-ccrMe was prepared.
- MF01 strain MF01 ⁇ B1 strain
- MF01 ⁇ B1B3 strain were each transformed.
- “phaJ4a” corresponds to the gene “phaJ1 Cn ” encoding (R) -specific enoyl-CoA hydratase described in International Publication WO2011 / 105379.
- crotonyl-CoA reductase is a bifunctional enzyme that exhibits not only reducing activity against crotonyl-CoA but also reductive carbon fixation in the presence of carbon dioxide (Erb, TJ, et al. Proc. Natl. Acad. Sci. US 106: 8871-8876 (2009)).
- This reductive carbonation reaction produces ethylmalonyl-CoA from crotonyl-CoA, which may constitute an undesirable alternative pathway in the P (3HB-co-3HHx) biosynthesis of the present invention.
- Example 3 C.I. PHA biosynthesis from fructose, glucose and glycerol by the nector NSDG ⁇ B-GG strain
- the necator wild strain H16 grows well with fructose and gluconic acid and accumulates polyester, but cannot grow with glucose and grows very slowly when glycerol is used as a carbon source.
- Glucose is a monosaccharide constituting starch and cellulose, and utilization of glucose is important from the viewpoint of utilization of plant-derived biomass resources.
- glycerol has been produced in large quantities as a by-product in the production of biodiesel from vegetable oil, and its effective use is desired.
- the NSDG ⁇ B-GG strain was prepared by accumulating in the nector NSDG strain and further deleting PhaB1, which is an acetoacetyl-CoA reductase. Using this strain as a host, a plasmid pBPP-ccr Me- phaJ4a-emd Mm for establishing a copolymer polyester biosynthetic pathway was introduced to biosynthesize copolyesters from various carbon sources.
- a region containing about 1 kbp each of the nagE gene and its upstream and downstream was amplified by PCR using the genomic DNA of necator H16 strain as a template and oligonucleotides of the following sequences 1 and 2 as primers.
- Sequence 15 GGAATTCATTGAGGTGGCCCGCGAATATCGGCAGCCT (SEQ ID NO: 19)
- Sequence 16 GGAATTCAGGTGCGCTTCGACAAGTCACTACTTT (SEQ ID NO: 20)
- the 5′-end of the amplified fragment was phosphorylated and inserted into the HincII site of the general-purpose plasmid pUC118.
- Sequence 17 GGCCAACCAGCGCGCGCCCCGCCGGCGGCGTCTCGT (SEQ ID NO: 21)
- Sequence 18 GCATGCCTGTTCTCGATGGCACTGACCT (SEQ ID NO: 22)
- the 5′-end of the amplified fragment was phosphorylated and self-ligated.
- the obtained plasmid was treated with restriction enzymes BamHI and XbaI to obtain a fragment containing a mutated nagE gene. This fragment was ligated with the fragment of pK18mobSacB cleaved with the same restriction enzymes to obtain pK18msNagE.
- G265R was obtained.
- the plasmid pK18ms ⁇ nagR for homologous recombination for deleting the nagR gene on the chromosome of the necator NSDG strain was prepared as follows. First, C.I. The region containing about 1 kbp each of the nagR gene and its upstream / downstream was amplified by PCR using the genomic DNA of necator H16 strain as a template and oligonucleotides of the following sequences 19 and 20 as primers.
- Sequence 19 TGCAGTTCGTATGCGACCGCATCGA (SEQ ID NO: 23)
- Sequence 20 GGAATTCAGGTGCGCTTCGACAAGTCCATACTTT (SEQ ID NO: 24)
- the 5′-end of the amplified fragment was phosphorylated and inserted into the HincII site of the general-purpose plasmid pUC118.
- a region not containing the nagR gene was amplified by an inverse PCR method using this plasmid as a template and oligonucleotides of the following sequences 21 and 22 as primers.
- Sequence 21 TGCCCGGCACGCCCCGCAACCGGGCGCTCGA (SEQ ID NO: 25)
- Sequence 22 TGCGAATCCCTCGTAGTACCAGATGTGGA (SEQ ID NO: 26)
- the 5′-end of the amplified fragment was phosphorylated and self-ligated.
- the obtained plasmid was treated with restriction enzymes EcoRI and HindIII to obtain a fragment in which the nagR gene was deleted and the upstream and downstream were connected.
- PK18ms ⁇ nagR was obtained by ligation with a fragment of pK18mobSacB cleaved with the same restriction enzyme.
- h16 by the PCR method using the genomic DNA of Necator H16 strain as a template and oligonucleotides of the following sequences 23 and 24 as primers.
- a region containing about 750 bp upstream and downstream of the start codon of the A2858 gene was amplified.
- Sequence 23 ATACCGTCGACGGGTGCTGGCTCCCGAGAGTTT (SEQ ID NO: 27)
- Sequence 24 CTGCAGTCGACCCTGCCGCGCCCACGCCGCTTTT (SEQ ID NO: 28)
- the amplified fragment was treated with the restriction enzyme SalI and inserted into the SalI site of pK18mobSacB.
- Sequence 25 GCGGGCAACGGATGGAGGTAAGCA (SEQ ID NO: 29)
- Sequence 26 CTTACCTCCATCCGTTGCCCGCTTCG (SEQ ID NO: 30)
- the glpFK gene region was amplified by PCR using the genomic DNA of Escherichia coli MG1655 strain as a template and oligonucleotides of the following sequences 27 and 28 as primers.
- Sequence 27 ATGAGTCAAAACATCAACCTT (SEQ ID NO: 31)
- Sequence 28 TTATTCGGTCGGTTCTCCCAC (SEQ ID NO: 32)
- the 5′-end of the amplified glpFK gene region fragment was phosphorylated and h16 was obtained by the above-described inverse PCR method. It was ligated with the fragment opened at the start codon of the A2858 gene.
- h16 The glpFK gene is h16 upstream of the A2858 gene.
- a plasmid ligated in the same direction as the A2858 gene was selected to obtain pK18msglpFK-A2858.
- Sequence 29 TCGACCGGCGCCGCACTTCTC (SEQ ID NO: 33)
- Sequence 30 GCATGCCAGTGTCTTACTTCT (SEQ ID NO: 34)
- the obtained DNA fragment was treated with restriction enzymes NdeI and SphI, and ligated with a fragment containing the plasmid backbone of pK18msNSDG-AB similarly cut with restriction enzymes NdeI and SphI to obtain pK18msC′R.
- C.I C.I.
- the ⁇ -ketothiolase gene phaA Cn having a recognition sequence for the restriction enzyme NdeI at both ends was amplified by PCR using the genomic DNA of necator H16 strain as a template and oligonucleotides of the following sequences 31 and 32 as primers.
- the obtained phaA Cn fragment was cleaved with NdeI and inserted into the NdeI site of pK18msC′R to obtain pK18msC′AR.
- This plasmid was digested with restriction enzymes SbfI and BamHI to remove the phaC NSDG gene, and blunt ended and self-ligated to obtain pK18msAR2.
- Necator NSDG ⁇ B-GG strain was prepared. Furthermore, it was introduced into NSDG ⁇ B-GG strain by conjugal transfer plasmid pBPP-ccr Me -phaJ4a-emd Mm for establishing a P (3HB-co-3HHx) biosynthetic pathway.
- PHA biosynthesis from glucose raw material, fructose raw material, and glycerol raw material is a nitrogen source-limited inorganic salt medium containing 1.0 wt% fructose, glucose, and glycerol as the sole carbon source. This was performed by culturing the cells. The culture time was 72 hours for fructose and glucose and 96 hours for glycerol. The accumulation rate and composition of PHA accumulated in the cells were determined by gas chromatography.
- Table 5 shows the results of culturing strains in which pBPP-ccrMeJ4a-emd was introduced into the nector NSDG ⁇ B-GG strain.
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Abstract
Description
[1]ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)生産能を付与した組換えCupriavidus necator(クプリアヴィダス・ネカトール)株の染色体に、クロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として糖質及び/又はグリセロールを含有する培地で形質転換体を増殖させることを含む、ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)を製造する方法。
[2]ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)生産能を付与した組換えC.necator株の染色体上のアセトアセチル-CoA還元酵素をコードする遺伝子を欠失させた組換え株の染色体に、クロトニル-CoA還元酵素遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として糖質及び/又はグリセロールを含有する培地で形質転換体を増殖させることを含む、ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)を製造する方法。
[3]前記形質転換体において、(R)-特異的エノイル-CoAヒドラターゼ遺伝子を相同性組換えによって形質転換し、又は該遺伝子が組み込まれた自律複製ベクターの導入によって形質転換することをさらに含む、上記[2]に記載の方法。
[4]前記形質転換体において、エチルマロニル-CoA脱炭酸酵素遺伝子を相同性組換えによって形質転換し、又は該遺伝子が組み込まれた自律複製ベクターの導入によって形質転換することをさらに含む、上記[2]及び[3]に記載の方法。
[5]C.necatorが、JMP134株(DSM4058)又はH16株(DSM428)である、上記[1]~[4]に記載の方法。
[6]組換えC.necator株が、MF01株、NSDG株、又はNSDGΔA株である、上記[1]~[5]に記載の方法。
[7]クロトニル-CoA還元酵素遺伝子が放線菌Streptomyces cinnamonensis(ストレプトミセス・シナモネンシス)由来である、上記[1]~[6]に記載の方法。
[8]クロトニル-CoA還元酵素遺伝子が、
(a)配列番号1で表される塩基配列を含む核酸;又は
(b)配列番号1で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつクロトニル-CoAからブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、上記[7]に記載の方法。
[9]クロトニル-CoA還元酵素遺伝子がメタノール資化性菌Methylobacterium extorquens(メチロバクテリウム・エクストークエンス)由来である、上記[1]~[6]に記載の方法。
[10]クロトニル-CoA還元酵素遺伝子が、
(a)配列番号2で表される塩基配列を含む核酸;又は
(b)配列番号2で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつクロトニル-CoAからブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、上記[9]に記載の方法。
[11](R)-特異的エノイル-CoAヒドラターゼ遺伝子が、C.necator由来であり、
(a)配列番号3で表される塩基配列を含む核酸;又は
(b)配列番号3で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化経路中間体である2-エノイル-CoAを(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、上記[1]~[10]に記載の方法。
[12]エチルマロニル-CoA脱炭酸酵素遺伝子が、
(a)配列番号4で表される塩基配列を含む核酸;又は
(b)配列番号4で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつエチルマロニル-CoAを脱炭酸し、ブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、上記[1]~[11]に記載の方法。
上記の通り、本発明者らは、アセチル-CoAから3HBユニットと3HHxユニットを生成し、共重合する新規な代謝経路を開拓することによって、従来の植物油とは異なり、広く糖質やグリセロールを出発原料として上記課題を解決することに成功した。より具体的には、本発明は、P(3HH-co-3HHx)生産能を付与した組換えC.necator株の染色体に、クロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として糖質やグリセロールを含有する培地で形質転換体を増殖させることを含む、P(3HH-co-3HHx)を製造する方法、並びに該共重合体の生産量及び/又は該共重合体中の3HHxユニットの分率を向上させる方法に関する。なお、ここで、クロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子は、そのすべてが相同性組換えによって形質転換されているか、又は前記株に該遺伝子が組み込まれた自律複製ベクターとして導入されていてもよいし、クロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子のうち、1つ又は2つの遺伝子が相同性組換えによって形質転換されていて、残りの遺伝子が前記株に該遺伝子が組み込まれた自律複製ベクターとして導入されている形態であってもかまわない。さらに、本態様において、C.necator株の染色体上のアセトアセチル-CoA還元酵素をコードする遺伝子が欠失されていてもよい。
本発明の生産方法に使用される宿主は、PHA生産菌であるC.necatorである。特に、本発明の生産方法に使用されるC.necatorとしては、限定されないが、JMP134株(DSM4058)及びH16株(DSM428)が挙げられる。より具体的には、本発明においては、P(3HB-co-3HHx)生産能を付与した組換えC.necator株を用いることが好ましく、例えば、NSDG株、MF01株、及びNSDGΔA株を使用してもよい。
本発明によれば、P(3HB-co-3HHx)の生産量の向上、及び/又は3HHx分率の向上には、該共重合体の生産能を付与した組換えC.necator株に形質転換によってクロトニル-CoA還元酵素をコードする遺伝子(ccr)を導入させることが必要である。ここで、本明細書において使用される「クロトニル-CoA還元酵素」とは、脂肪酸β-酸化経路の中間体である炭素数4のクロトニル-CoAを還元し、β-ケトチオラーゼ(BktB)の基質となるブチリル-CoAを生成する酵素である。ブチリル-CoAがβ-ケトチオラーゼの作用によりもう1分子のアセチル-CoAと縮合し、さらに変換されることにより炭素数6の(R)-3HHx-CoAが供給され、広基質特異性を示すポリエステル重合酵素により(R)-3HB-CoAとともに共重合される。本発明において使用され得るccrは、翻訳後の該還元酵素が上記の活性を有する限り、生物種の由来は特に限定されないが、好ましくは、放線菌S.cinnamonensis由来のクロトニル-CoA還元酵素をコードする遺伝子(以下「ccrSc」と称することがある)又はメタノール資化性菌M.extorquens由来のクロトニル-CoA還元酵素をコードする遺伝子(以下「ccrMe」と称することがある)である。
本発明による共重合体の生産方法において、上記のクロトニル-CoA還元酵素をコード遺伝子が導入された宿主に、(R)-特異的エノイル-CoAヒドラターゼをコードする遺伝子(phaJ)をさらに導入してもよい。ここで、本発明に使用される「(R)-特異的エノイル-CoAヒドラターゼ」とは、脂肪酸β-酸化系中間体である2-エノイル-CoAをPHAモノマーである(R)-3-ヒドロキシアシル-CoAに変換する酵素を意味し、この活性を有する限りにおいては、生物種の由来は特に限定されないが、好ましくは、C.necator株由来である。
本発明による共重合体の生産方法において、上記のクロトニル-CoA還元酵素をコード遺伝子が導入された宿主に、(R)-特異的エノイル-CoAヒドラターゼをコードする遺伝子(phaJ)に代えて又は該遺伝子に加えて、エチルマロニル-CoA脱炭酸酵素遺伝子(emd)を導入してもよい。ここで、本発明に使用される「エチルマロニル-CoA脱炭酸酵素」とは、動物細胞においてプロピオニル-CoAカルボキシラーゼなどによる副反応で生じたエチルマロニル-CoAのブチリル-CoAへの脱炭酸反応を触媒する酵素を意味し、この活性を有する限りにおいては、生物種の由来は特に限定されない。
本発明の一態様によれば、アセトアセチル-CoA還元酵素をコードする遺伝子を欠失させた組換えC.necator株を用いることが好ましい。「アセトアセチル-CoA還元酵素」とは、アセトアセチル-CoAを基質として(R)-3HB-CoAを生成する触媒機能を有する酵素であり、C.necatorにおいては、「PhaB1」、「PhaB2」、及び「PhaB3」が知られ、フルクトースでの増殖条件における上記の反応においては、PhaB1が主体となって機能するが、パラログであるPhaB3も機能することが報告されている(Budde,C.F.,et al.,J.Bacteriol.,192:5319-5328(2010))。上記の通り、本発明の一態様の生産方法に使用される組換えC.necator株として、アセトアセチル-CoA還元酵素をコードする遺伝子を欠失させた株を使用することが好ましく、欠失させるアセトアセチル-CoA還元酵素をコード遺伝子としては、好ましくはphaB1又はphaB1及びphaB3であり、より好ましくはphaB1である。ここで、欠失させる遺伝子(phaB1とphaB3)の塩基配列は公知であり、例えば、それぞれNCBI-GeneID:4249784、NCBI-GeneID:4250155を参照することによって、これらの遺伝子のいずれか又はその両方を欠失させた組換えC.necator株を得るため利用することができる。本明細書において使用するとき、「欠失」とは、対象とする遺伝子の一部又は全部が、遺伝子操作によって存在しなくなった状態を意味し、その結果として、該遺伝子によってコードされたタンパク質の活性の一部又は全部が失われることを意図する。なお、欠失の変異は、公知の部位突然変異誘発方法(Current Protocols in Molecular Biology 1巻,8.1.1頁,1994年)により、あるいは市販のキット(Takara社のLA PCR in vitro Mutagenesisシリーズキット)を用いて誘発することができる。
本発明によれば、ccr、phaJ、及びemdを宿主の染色体に導入するための、これらの遺伝子の各々若しくはいずれかの組み合わせを相同性組換え用ベクターに組み込んだ遺伝子置換ベクター、又は該遺伝子の各々若しくはいずれかの組み合わせを自律複製ベクターに組み込んだ発現ベクターが提供される。ここで、ベクターに遺伝子を組み込む方法としては、例えば、Sambrook,J.ら,Molecular Cloning,A Laboratory Manual(3rd edition),Cold Spring Harbor Laboratory,1.1(2001)に記載の方法などが挙げられる。簡便には、市販のライゲーションキット(例えば、トーヨーボー社製等)を用いることもできる。
本発明によれば、P(3HB-co-3HHx)共重合体の合成は、該共重合体生産能を付与した組換えC.necator株の染色体にクロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子を導入することによって、又は該共重合体生産能を付与した組換えC.necator株のアセトアセチル-CoA還元酵素をコードする遺伝子を欠失させた組換え株の染色体にクロトニル-CoA還元酵素遺伝子を導入し、さらには(R)-特異的エノイル-CoAヒドラターゼ遺伝子又はエチルマロニル-CoA脱炭酸酵素遺伝子を導入することによって、該組換え株内又は培養物(例えば、培地)中に該共重合体を生成及び蓄積させ、組換え株又は培養物から目的とする該共重合体を採取することにより行われる。なお、当業者にも理解されるように、該共重合体を合成させるために、上記組換え株を適切な培養条件下に置くことが好ましい。このような組換え株の培養、遺伝子組換えを行う前の親株の培養条件に従ってもよい。また、本発明の特定の一実施形態において、炭素源として糖質及び/又はグリセロールを含有する培地中で組換え株を増殖させてもよい。
本発明において、共重合体は、下記の通り精製することができる:培地から遠心分離によって形質転換体を回収し、蒸留水で洗浄後、乾燥又は凍結乾燥させる。その後、クロロホルムに乾燥した形質転換体を懸濁させ、室温で所定時間撹拌し、共重合体を抽出する。抽出の段階で、必要であれば加熱してもよい。濾過によって残渣を除去し、上清にメタノールを加えて共重合体を沈殿させ、沈殿物を濾過又は遠心分離によって、上清を除去し、乾燥させて精製した共重合体を得ることができる。その後、限定されないが、NMR(核磁気共鳴)、ガスクロマトグラフィーを用いて、得られた共重合体のモノマーユニットの組成比を確認することができる。
(1)宿主
C.necator野生株であるH16株は、P(3HB)生合成経路を構成するPHA重合酵素(PhaC)、β-ケトチオラーゼ(PhaA)、アセトアセチル-CoA還元酵素(PhaB1)をコードするphaCAB1オペロンを染色体上に有する。以前に作製したC.necator MF01株は、H16株のphaCAB1オペロン中のphaCをA.caviae由来のPHA重合酵素の変異遺伝子phaCNSDGに、phaAをC.necator由来の広基質特異性β-ケトチオラーゼの遺伝子bktBに置換した組換え株であり(図1)、大豆油を炭素源として2.6mol%の3HHxユニットを含むP(3HB-co-3HHx)を生合成可能である(国際公開WO2011/105379、Mifune,J.,et al.,Polym.Degrad.Stab.,95:1305-1312(2010))。以下、これらの株を用いて、P(3HB-co-3HHx)の生産量及び3HHx分率について検討した。なお、以下の実施例において、PCRは基本的にKOD Plus DNAポリメラーゼ(トーヨーボー社製)を用い、98℃で20秒、60℃で20秒、68℃で2分30秒の反応を1サイクルとしてこれを30サイクル行い、必要に応じて温度条件を適宜、調節した。
C.necator MF01株の染色体上でアセトアセチル-CoA還元酵素PhaB1をコードする遺伝子(phaB1)を欠失させるための相同性組換え用プラスミドpK18msbktBRは以下のように作製した。まず、C.necator H16株のゲノムDNA断片を鋳型として下記の配列1と配列2のオリゴヌクレオチドをプライマーとしたPCR法によって、C.necator由来のβ-ケトチオラーゼ(BktB)を増幅した。
配列1:TACATGGATCCAAGGGAGGCAAAGTCATGACGCGTGAAGTGGTAGTG(配列番号5)
配列2:GGATCATATGCTTCCTCAGATACGCTCGAAGATGGC(配列番号6)
増幅したbktB断片を制限酵素BamHIとNdeIで処理した。この制限酵素処理断片を、以前に作製された相同性組換え用プラスミドpK18msNSDG-R(phaCNSDGとphaB1下流領域が連結されたDNA断片を含む)(Mifune,J.,et al.,Polym.Degrad.Stab.,95:1305-1312(2010))を制限酵素BamHIとNdeIで処理した断片と連結することによりphaB1欠失用プラスミドpK18msbktBRを得た。
C.necator MF01株の染色体上でアセトアセチル-CoA還元酵素PhaB3をコードする遺伝子(phaB3)を欠失させるための相同性組換え用プラスミドpK18msΔphaB3は以下のように作製した。まず、C.necator H16株のゲノムDNA断片を鋳型として下記の配列3と配列4、及び配列5と配列6のオリゴヌクレオチドをプライマーとしたPCR法によって、C.necator株由来のphaB3の上流領域と下流領域をそれぞれ増幅した。
配列3:TTTGGAATTCTACCTAGGGATCAAATTAGAGGAAA(配列番号7)
配列4:CCTTACTGCATGTGCCTGCTTCATTCTCGTAAAGTTGAAAG(配列番号8)
配列5:GAATGAAGCAGGCACATGCAGTAAGGGTGCTGGG(配列番号9)
配列6:TCCTAAGCTTGCTGACCGTGATCGTCGACAACTTTGAAGACCTGA(配列番号10)
ここで、配列4と配列5には互いにオーバーラップする領域を付加してある。次に、増幅したphaB3の上流断片と下流断片を精製して混合し、配列3と配列6のオリゴヌクレオチドをプライマーとしたフュージョンPCR法によってphaB3の上下流が連結された断片を増幅した。増幅したphaB3の上下流連結断片をEcoRIとHindIIIで処理した。この制限酵素処理断片を、EcoRIとHindIIIで処理したベクタープラスミドpK18mobsacBと連結することによりphaB3欠失用プラスミドpK18msΔphaB3を得た。
実施例1(2)で得られた組換えプラスミドpK18msbktBR、(3)で得られたpK18msΔphaB3を、C.necator MF01株に接合伝達により導入し、相同性組換えによって遺伝子が破壊された株を取得した。まず、塩化カルシウム法によって、作製したベクターを大腸菌S17-1株に導入した。次に、この組換え大腸菌をLB培地(1%トリプトン、1%塩化ナトリウム、0.5%イーストエキス、pH7.2)3.0ml中で37℃終夜培養した。これと並行して、C.necator MF01株をNR培地(1%魚肉エキス、1%ポリペプトン、0.2%イーストエキス)3.0ml中で30℃終夜培養した。その後、大腸菌の培養液0.2mlに対して、C.necator MF01株の培養液0.1mlを混合し、30℃で6時間培養した。この菌体混合液を0.2mg/mlカナマイシンを添加したSimmons Citrate寒天培地(ディフコ社製)に塗布し、30℃で3日間培養した。組換え大腸菌のプラスミドが、C.necatorに伝達され相同性組換えにより染色体上に取り込まれた該菌体はカナマイシン耐性を示し、一方、組換え大腸菌はSimmons Citrate寒天培地では増殖不能であるため、上記培地上で増殖したコロニーは組換え大腸菌からベクターが染色体に取り込まれたC.necator形質転換体(ポップイン株)である。さらに、ポップイン株をNR培地で30℃終夜培養した後、10%スクロースを添加したNR培地に塗布し、30℃で3日間培養した。pK18mobsacB由来ベクター上のsacBにコードされるレヴァンスクラーゼはスクロースを基質にして細胞内に毒性多糖を蓄積する。このため、10%スクロース添加培地においてはプラスミド領域が脱離した株(ポップアウト株)のみが生育することができる。これらのコロニーの中から染色体上において標的部位での相同性組換えが生じた株をPCR法によって選抜した。pK18msbktBRを用いてMF01株を形質転換することでphaB1が欠失したMF01ΔB1株を、pK18msΔphaB3を用いてMF01株を形質転換することでphaB3が欠失したMF01ΔB3株を取得した。さらにpK18msΔphaB3を用いてMF01ΔB1株を形質転換することでphaB1とphaB3が二重欠失したMF01ΔΔB1B3株を取得した(図1)。
放線菌S.cinnamonensis由来クロトニル-CoA還元酵素遺伝子(ccrSc)を、C.necator細胞内で自律複製可能なプラスミドpBBR1-MCS2のlacプロモーター下流に配した発現ベクター、pBBR-ccrScを作製した。また、メタノール資化性菌M.extorquens由来クロトニル-CoA還元酵素遺伝子(ccrMe)をC.necator細胞内で自律複製可能なプラスミドpBBR1-MCS2のlacプロモーター下流に配した発現ベクター、pBBR-ccrMe、及びpBPPのphaP1プロモーター下流に廃した発現ベクター、pBPP-ccrMeを作製した。より具体的には、以下の通りである。
ccrScがすでに組み込まれたpJBccrEE32d13(Fukui,T.,et al.,Biomacromolecules,3:618-624(2002))を鋳型として下記の配列7と配列8のオリゴヌクレオチドをプライマーとしたPCR法によって、ccrScを増幅した。なお、ccrSc(配列番号1)はGTGを開始コドンとするが、配列7を用いたPCRによって開始コドンはATGに変換される。
配列7:ACGAATTCAGGAGGAACCTGGATGAAGGAAATCCTGGACG(配列番号11)
配列8:AGGTCTAGAGTGCGTTCAGACGTTGCGGA(配列番号12)
増幅したccrSc断片を制限酵素EcoRIとXbaIで処理した。この制限酵素処理断片を、pBBR1-MCS2を制限酵素EcoRIとXbaIで処理した断片と連結することによりccrSc発現用プラスミドpBBR-ccrScを得た。
M.extorquens AM1株のゲノムDNAを鋳型として下記の配列9と配列10のオリゴヌクレオチドをプライマーとしたPCR法によって、ccrMeを増幅した。
配列9:ACGAATTCAGGAGGAACCTGGATGGCTGCAAGCGCAGCACC(配列番号13)
配列10:AGGTCTAGATCACATCGCCTTGAGCGG(配列番号14)
増幅したccrMe断片を制限酵素EcoRIとXbaIで処理した。この制限酵素処理断片を、pBBR1-MCS2を制限酵素EcoRIとXbaIで処理した断片と連結することによりccrMe発現用プラスミドpBBR-ccrMeを得た。
M.extorquens AM1株のゲノムDNAを鋳型として下記の配列9と配列10のオリゴヌクレオチドをプライマーとしたPCR法によって、ccrMeを増幅した。
配列11:ATACATATGGCTGCAAGCGCAGCACCGGCCT(配列番号15)
配列12:TATGAATTCTCACATCGCCTTGAGCGGGCC(配列番号16)
増幅したccrMe断片を制限酵素NdeIとEcoRIで処理した。この制限酵素処理断片を、pBPPを制限酵素NdeIとEcoRIで処理した断片と連結することによりccrMe発現用プラスミドpBPP-ccrMe(図2)を得た。
C.necator H161株のゲノムDNAを鋳型として下記の配列11と配列12のオリゴヌクレオチドをプライマーとしたPCR法によって、phaJ4aを増幅した。
配列13:CCCAAGCTTTATCGTCAAGAGGAGACTATCG(配列番号17)
配列14:CCCAAGCTTGGATCCTCACCCGTAGCGGCGCGTGAT(配列番号18)
増幅したphaJ4a断片を制限酵素HindIIIで処理した。この制限酵素処理断片を、pBPP-ccrMeを制限酵素HindIIIで処理した断片と連結することによりccrMe及びphaJ4a共発現用プラスミドpBPP-ccrMeJ4a(図2)を得た。
まず、pBPP-ccrMeJ4aをEcoRIとHindIIIで切断し、末端平滑化後にセルフライゲーションすることで、ベクター上のEcoRIサイトからHindIIIサイトまでに領域が欠失したプラスミドを作製した。人工合成したemdMmが組み込まれたベクター(オペロン社により委託合成)からemdMmをBamHIで切り出し、上述のpBPP-ccrMeJ4a改変ベクターをBamHIで処理した断片と連結することにより、ccrMe、phaJ4a、emdMm共発現用プラスミドpBPP-ccrMeJ4a-emd(図2)を得た。
C.necator MF01株(図1)を宿主とし、pBBR1-ccrSc又はpBBR1-ccrMeを導入した組換え株を0.5%フルクトースを唯一の炭素源とする窒素源制限無機塩培地で培養したところ、いずれの株も蓄積PHAはP(3HB)であり、3HHxユニットは検出されなかった(表1)。
上述のように外来ccrの発現はPHA生産量の減少を伴うため、P(3HB-co-3HHx)生合成経路の改良を行った。(R)-3HB-CoA及び(R)-3HHx-CoAの両方の供給の強化を目的として、C.necator由来(R)-特異的エノイル-CoAヒドラターゼの1つをコードする遺伝子(phaJ4a)を使用した。その発現産物であるPhaJ4aは、C4、C6のエノイル-CoAに対して(R)-特異的な水和反応を触媒することで(R)-3HB-CoA、(R)-3HHx-CoAを生成するが、C4よりC6のエノイル-CoAに対して高い活性を示すことが報告されている(国際公開WO2011/105379;Kawashima,Y.,et al.,Appl.Environ.Microbiol.,78:493-502(2012))。pBPP-ccrMeにphaJ4aを挿入した発現ベクターpBPP-ccrMeJ4a(図2)を作製し、C.necator MF01株、MF01ΔB1株、MF01ΔΔB1B3株をそれぞれ形質転換した。なお、本明細書中の「phaJ4a」は国際公開WO2011/105379に記載の(R)-特異的エノイル-CoAヒドラターゼをコードする遺伝子「phaJ1Cn」に対応する。
C.necator野生株であるH16株はフルクトースやグルコン酸で良好に増殖しポリエステルを蓄積するが、グルコースでは増殖できず、またグリセロールを炭素源とした際の増殖は極めて遅い。グルコースはデンプンやセルロースを構成する単糖であり、グルコースの利用は植物由来バイオマス資源の利用の観点で重要である。また、グリセロールは近年、植物油からのバイオディーゼルの生産における副成生物として大量に生じており、その有効利用が望まれている。
(1)nagE変異及びnagR欠失用プラスミドベクターの作製
C.necator NSDG株へのグルコース資化能の付与はNagEの265番目グリシンからアルギニンへの置換、及びNagRの欠失により行った。NagEのアミノ酸置換は染色体上のnagE遺伝子793番目の塩基をグアニンからシトシンに置換する変異により導入した。このための相同性組換え用プラスミドpK18msNagE G265Rは以下のように作製した。まず、C.necator H16株のゲノムDNAを鋳型、下記の配列1と配列2のオリゴヌクレオチドをプライマーとしたPCR法によって、nagE遺伝子とその上下流それぞれ約1kbpを含む領域を増幅した。
配列15:GGAATTCTATTGAGGTGGCCGCGAATATCGGCAGCCT(配列番号19)
配列16:GGAATTCAGGTGCGCTTCGACAAGTCATACTTT(配列番号20)
増幅した断片の5’-末端をリン酸化し、汎用プラスミドpUC118のHincII部位に挿入した。このプラスミドを鋳型、下記の配列17と配列18のオリゴヌクレオチドをプライマーとしたインバースPCR法によって、nagE遺伝子の793番目塩基のグアニンをシトシンに置換する変異を導入した。
配列17:GGCCAACCAGCGCGCGCCCCGCCGGCGGCGTCTCGT(配列番号21)
配列18:GCATGCTGTTCTCGATGGCACTGACCT(配列番号22)
増幅した断片の5’-末端をリン酸化し、セルフライゲーションした。得られたプラスミドを制限酵素BamHIとXbaIで処理し、変異nagE遺伝子を含む断片を得た。この断片を、同じ制限酵素で切断したpK18mobSacBの断片と連結することによりpK18msNagE G265Rを得た。
配列19:TGCAGTTCGTATGCGACCGCATCGA(配列番号23)
配列20:GGAATTCAGGTGCGCTTCGACAAGTCATACTTT(配列番号24)
増幅した断片の5’-末端をリン酸化し、汎用プラスミドpUC118のHincII部位に挿入した。このプラスミドを鋳型、下記の配列21と配列22のオリゴヌクレオチドをプライマーとしたインバースPCR法によって、nagR遺伝子を含まない領域を増幅した。
配列21:TGCCCGGCACGCCCGGCAACCGGCGGCTCGA(配列番号25)
配列22:TGCGAATCCTCGTAGGTACCAGAGTGTGGA(配列番号26)
増幅した断片の5’-末端をリン酸化し、セルフライゲーションした。得られたプラスミドを制限酵素EcoRIとHindIIIで処理し、nagR遺伝子が欠失して上下流が連結された断片を得た。同じ制限酵素で切断したpK18mobSacBの断片と連結することによりpK18msΔnagRを得た。
C.necator NSDG株のグリセロール資化能の強化は、大腸菌由来のglpF-glpK(以下、glpFKとする)遺伝子をC.necator NSDG株の染色体上の機能未知遺伝子であるh16 A2858の上流に挿入することで行った。このための相同性組換え用プラスミドpK18msglpFK-A2858は以下のように作製した。まず、C.necator H16株のゲノムDNAを鋳型、下記の配列23と配列24のオリゴヌクレオチドをプライマーとしたPCR法によって、h16 A2858遺伝子の開始コドンの上下流それぞれ約750bpを含む領域を増幅した。
配列23:ATACCGTCGACGGTGCTGGCTCCGGAAGGTTT(配列番号27)
配列24:CTGCAGTCGACCCTGCGCGCCCACGCCGCTTT(配列番号28)
増幅した断片を制限酵素SalIで処理し、pK18mobSacBのSalI部位に挿入した。得られたプラスミドを鋳型、下記の配列25と配列26のオリゴヌクレオチドをプライマーとしたインバースPCR法によって、h16 A2858遺伝子の開始コドンで開環した断片を増幅した
配列25:GCGGGCAACGGATGGAGGTAAGCA(配列番号29)
配列26:CTTACCTCCATCCGTTGCCCGCTTCG(配列番号30)
配列27:ATGAGTCAAACATCAACCTT(配列番号31)
配列28:TTATTCGTCGTGTTCTTCCCAC(配列番号32)
増幅したglpFK遺伝子領域断片の5’-末端をリン酸化し、上記のインバースPCR法によってh16 A2858遺伝子の開始コドンで開環した断片と連結した。h16 A2858遺伝子の上流にglpFK遺伝子がh16 A2858遺伝子と同じ向きに連結されたプラスミドを選抜し、pK18msglpFK-A2858を得た。
C.necator NSDG株染色体上のphaオペロンからアセトアセチル-CoA還元酵素1をコードするphaB1Cn遺伝子を破壊するためのプラスミドpK18msAR2は、以前に作製したpK18msNSDG-AB(WO2011/105379)を基に、以下のように作製した。まずC.necator H16株のゲノムDNAをテンプレート、下記の配列29と配列30のオリゴヌクレオチドをプライマーとしたPCR法によってphaB1Cn遺伝子下流の領域約1kbpを増幅した。
配列29:TCGACCGGCGCCGACTTCTC(配列番号33)
配列30:GCATGCCAGTGTCTTACTTCT(配列番号34)
得られたDNA断片を制限酵素NdeI及びSphIで処理し、同様に制限酵素NdeI及びSphIで切断したpK18msNSDG-ABのプラスミド骨格を含む断片と連結することでpK18msC’Rを得た。また、C.necator H16株のゲノムDNAをテンプレート、下記の配列31と配列32のオリゴヌクレオチドをプライマーとしたPCR法によって、制限酵素NdeIの認識配列を両末端に有するβ-ケトチオラーゼ遺伝子phaACnを増幅した。
配列31:CGCCGCATGACGCTTGCATA(配列番号35)
配列32:CCATATGCGGCCCCGGAAAACCCC(配列番号36)
得られたphaACn断片をNdeIで切断し、pK18msC’RのNdeI部位に挿入することでpK18msC’ARを得た。このプラスミドを制限酵素SbfI及びBamHIで切断することでphaCNSDG遺伝子を除去し、平滑末端化の後にセルフライゲーションを行うことでpK18msAR2を得た。
上記で作製したpK18mobSacBベースの相同性組換え用ベクターのC.necatorへの導入、及び相同性組換え株の選抜は実施例1と同様の方法で行った。NSDG株を初発の宿主とし、pK18msNagE G265R、pK18msΔnagR、pK18msglpFK-A2858、pK18msAR2を順次、接合伝達により導入して目的とする相同性組換え株を選抜することで、グルコース資化能付与・グリセロール資化能強化・PhaB1Cn欠失のC.necator NSDGΔB-GG株を作製した。さらに、P(3HB-co-3HHx)生合成経路を確立するためのプラスミドpBPP-ccrMe-phaJ4a-emdMmを接合伝達によりNSDGΔB-GG株に導入した。
作製した組換え株によるPHA生合成は1.0重量%のフルクトース、グルコース、グリセロールを唯一の炭素源とする窒素源制限無機塩培地で培養することにより行った。培養時間はフルクトース、グルコースの場合は72時間、グリセロールの場合は96時間とした。菌体内に蓄積されたPHAはガスクロマトグラフ法により蓄積率及び組成を決定した。
Claims (12)
- ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)生産能を付与した組換えCupriavidus necator(クプリアヴィダス・ネカトール)株の染色体に、クロトニル-CoA還元酵素遺伝子、(R)-特異的エノイル-CoAヒドラターゼ遺伝子、及びエチルマロニル-CoA脱炭酸酵素遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として糖質及び/又はグリセロールを含有する培地で形質転換体を増殖させることを含む、ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)を製造する方法。
- ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)生産能を付与した組換えC.necator株の染色体上のアセトアセチル-CoA還元酵素をコードする遺伝子を欠失させた組換え株の染色体に、クロトニル-CoA還元酵素遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として糖質及び/又はグリセロールを含有する培地で形質転換体を増殖させることを含む、ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)を製造する方法。
- 前記形質転換体において、(R)-特異的エノイルCoAヒドラターゼ遺伝子を相同性組換えによって形質転換し、又は該遺伝子が組み込まれた自律複製ベクターの導入によって形質転換することをさらに含む、請求項2に記載の方法。
- 前記形質転換体において、エチルマロニル-CoA脱炭酸酵素遺伝子を相同性組換えによって形質転換し、又は該遺伝子が組み込まれた自律複製ベクターの導入によって形質転換することをさらに含む、請求項2又は3に記載の方法。
- C.necatorが、JMP134株(DSM4058)又はH16株(DSM428)である、請求項1~4のいずれか1項に記載の方法。
- 組換えC.necator株が、MF01株、NSDG株、又はNSDGΔA株である、請求項1~5のいずれか1項に記載の方法。
- クロトニル-CoA還元酵素遺伝子が放線菌Streptomyces cinnamonensis(ストレプトミセス・シナモネンシス)由来である、請求項1~6のいずれか1項に記載の方法。
- クロトニル-CoA還元酵素遺伝子が、
(a)配列番号1で表される塩基配列を含む核酸;又は
(b)配列番号1で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつクロトニル-CoAからブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、請求項7に記載の方法。 - クロトニル-CoA還元酵素遺伝子がメタノール資化性菌Methylobacterium extorquens(メチロバクテリウム・エクストークエンス)由来である、請求項1~6のいずれか1項に記載の方法。
- クロトニル-CoA還元酵素遺伝子が、
(a)配列番号2で表される塩基配列を含む核酸;又は
(b)配列番号2で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつクロトニル-CoAからブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、請求項9に記載の方法。 - (R)-特異的エノイル-CoAヒドラターゼ遺伝子が、C.necator由来であり、
(a)配列番号3で表される塩基配列を含む核酸;又は
(b)配列番号3で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化経路中間体である2-エノイル-CoAを(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1~10のいずれか1項に記載の方法。 - エチルマロニル-CoA脱炭酸酵素遺伝子が、
(a)配列番号4で表される塩基配列を含む核酸;又は
(b)配列番号4で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつエチルマロニル-CoAを脱炭酸し、ブチリル-CoAを生成する触媒活性を有するタンパク質をコードする核酸
からなる、請求項1~11のいずれか1項に記載の方法。
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JP6755515B2 (ja) | 2020-09-16 |
CN106574279A (zh) | 2017-04-19 |
EP3187590B1 (en) | 2020-02-26 |
JPWO2016021604A1 (ja) | 2017-05-18 |
EP3187590A1 (en) | 2017-07-05 |
US10538791B2 (en) | 2020-01-21 |
EP3187590A4 (en) | 2018-01-24 |
US20170218411A1 (en) | 2017-08-03 |
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