WO2020218566A1 - 高分子量コポリマーを合成するための遺伝子 - Google Patents
高分子量コポリマーを合成するための遺伝子 Download PDFInfo
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Definitions
- the present invention relates to a gene encoding a polymer synthase derived from a mangrove soil metagenome and a gene encoding an enoyl-CoA hydratase derived from Streptomyces species CFMR7 (accession number: CP011522).
- the present invention details the functional genes encoding ultra-high molecular weight copolymers and enoyl-CoA hydratase, which, when used together, result in the synthesis of high molecular weight copolymers.
- PHAs Polyhydroxyalkanoates
- bacteria and archaea storage compounds
- storage compounds reserve carbon
- PHA synthase An important enzyme involved in the polymerization of PHA is PHA synthase (PhaC). PhaC is an interesting enzyme because it can polymerize high molecular weight hydrophobic PHA chains in a cytoplasmic hydrophilic environment.
- PHA producers and PhaCs are primarily derived from studies of pure microbial isolates using a culture-dependent approach.
- a total of four PhC and 167 PHA producers based on the genus have been reported from existing culturable soil microorganisms, the existing culturable soil microorganisms being considered to be less than 15% of all soil microorganisms. .. The remaining 85% have not yet been investigated. Therefore, there is a great gap in understanding the diversity of PHA producers from this pool of unstudied microbial communities.
- the mangrove soil biome contains a high microbial diversity and is continuously exposed to various abiotic stresses such as saline and anoxic conditions.
- Several studies have reported the separation of PHA producers from the mangrove environment.
- no studies have been reported on PhaC derived from mangrove soil metagenomics. Therefore, it is likely that a large number of novel PhaCs will be discovered in new microbial genera in the mangrove soil metagenomics, especially in anaerobic microorganisms that are difficult to culture in the laboratory.
- Bacterial PHA can be divided into three major types depending on the number of carbon atoms in the monomer unit, such as the combination of short chain length (scl), medium chain length (mcl) and scl-mcl. .. scl-PHA consists of 3 to 5 carbon atoms and mcl-PHA has 6 to 14 carbon atoms, but the number of carbon atoms in scl-mcl-PHA ranges from 3 to 14 per monomer. possible.
- the type of PHA produced depends on the substrate specificity of the polymer synthase.
- PHAs consisting primarily of scl monomers are often hard and brittle, while PHAs consisting primarily of mcl monomers are elastic in nature.
- the Scl-mcl PHA copolymer can have properties between the two states, depending on the ratio of the scl and mcl monomers in the copolymer.
- the enoyl-CoA hydratase encoded by phaJ is a (R) -3-hydroxyacyl-CoA monomer unit during biosynthesis, eg, 3-hydroxyhexanoate coenzyme A (3HHx-CoA) from fatty acid ⁇ -oxidation. Shows (R) specific hydratase activity that feeds a poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P (3HB-co-3HHx)] copolymer.
- Patent Document 1 (US Pat. No. 6,812,013) describes a PHA synthase useful in the process of preparing PHA, a gene encoding the enzyme, a recombinant vector containing the gene, and a trait transformed by the vector. It relates to a transformant, a process for producing a PHA synthase using the transformant, and a process for preparing PHA using the transformant.
- a feature of the present invention is that it is a transformant in which a PSA synthase gene derived from Pseudomonas putida is introduced into a host microorganism that is cultured to produce PHA synthase or PHA.
- Patent Document 2 (US Pat. No. 2004-146998) relates to a transformant and a polymer production process using the transformant.
- the present invention discloses a gene encoding a copolymer synthase, a microorganism for fermentation synthesis of a polymer using the gene, and a method for producing a polymer by the microorganism.
- the present invention focuses on the construction of transformants which contain polyester synthesis-related enzyme genes, promoters and terminators and have been introduced into yeast.
- Patent Document 3 European Patent No. 1626087.
- the present invention discloses a gene expression cassette containing a gene encoding a PHA synthase derived from Aeromonas caviae. Yeast is also used as a host and mutations have been introduced into promoters and terminators, allowing gene cassettes to function within yeast.
- Patent Document 4 discloses transformants and processes for producing gene expression products in yeast. The transformant is obtained by introducing a plurality of enzyme genes involved in PHA synthesis, for example, a combination of PHA synthase and acetoacetyl-CoA reductase gene.
- Patent Document 5 discloses a recombinant microorganism that expresses both PHA synthase and intracellular PHA depolymerizer. The present invention is an invention that enables simultaneous synthesis and decomposition of PHA.
- Patent Document 6 (US Pat. No. 2012-808280) describes a novel polymer synthase derived from the genus Chromobacterium, a gene encoding the enzyme, a recombinant vector containing the gene, and transformation transformed by the vector. The body and the process of producing a plastic polymer using this transformant are disclosed.
- a main object of the present invention is to provide a polymer synthase gene derived from a mangrove soil metagenome and a method for producing a useful copolymer using this polymer synthase.
- Another object of the present invention provides a method for producing an enoyl-CoA hydratase gene derived from Streptomyces species CFMR7 and a useful copolymer P (3HB-co-3HHx) in which the composition of 3HHx is increased by the expression of the enoyl-CoA hydratase. It is to be.
- Yet another object of the present invention is to develop a method for more efficiently producing a higher molecular weight 3HHx copolymer by using a transformant containing a polymer synthase.
- Yet another object of the present invention is to develop a method for more efficiently producing a copolymer having a lipase-degradable monomer sequence such as 4HB and 5HV by using a transformant containing a polymer synthase. ..
- the present invention comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted or added, and having polymer synthase activity.
- the isolated polypeptide encoding is disclosed.
- the isolated polynucleotide is the nucleotide sequence set forth in SEQ ID NO: 2, or the nucleotide sequence set forth in SEQ ID NO: 2 in which one or more nucleotides are substituted. Or a complementary sequence thereof.
- the nucleotide sequence set forth in SEQ ID NO: 2 includes a nucleotide sequence in which thymine is replaced by uracil or a complementary sequence thereof.
- Another invention encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted or added, and having hydratase activity. Disclose the isolated polynucleotides to be used.
- nucleotide sequence set forth in SEQ ID NO: 4 or the nucleotide sequence set forth in SEQ ID NO: 2 in which one or more nucleotides are substituted, or a complementary sequence thereof is included.
- the nucleotide sequence set forth in SEQ ID NO: 4 includes a nucleotide sequence in which thymine is replaced by uracil, or a complementary sequence thereof.
- Yet another embodiment of the present invention is a recombinant vector containing the polynucleotide described in the previous embodiment.
- the recombinant vector is a plasmid or phage.
- a further embodiment of the present invention is a recombinant strain having the polynucleotide described in the previous embodiment in its genome.
- Another further embodiment of the invention comprises culturing the transformant or recombinant of the previous embodiment in a medium containing a polymerizable material; and recovering the polymer from the cultured medium.
- This is a method for producing a polymer.
- the polymer is PHA.
- the present invention relates to a gene encoding a polymer synthase derived from a mangrove soil metagenome and a gene encoding an enoyl-CoA hydratase derived from Streptomyces species CFMR7. More specifically, the present invention comprises recombinant vectors having these functional genes encoding polymer synthase and enoyl-CoA hydratase, respectively, the polymer synthase gene and / or enoyl-CoA hydratase alone, or both.
- the transformed strains that are expressed and the process of producing polymers via these functional genes will be described in detail.
- the present invention describes a novel PhaC [PhaC BP-M-CPF4 (accession number: AXB72506)] with extremely broad substrate specificity found in Barik Pulau (Penang, Malaysia) mangrove soil.
- the PhaC is scl-PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymer [P (3HB-co-3HV)], poly (3-hydroxybutyrate-co-3-).
- P (3HB-co-3H4MV) poly (3-hydroxybutyrate-co-4-hydroxybutyrate) random copolymer [P (3HB-co-4HB)] and A poly (3-hydroxybutyrate-co-5-hydroxyvalerate) random copolymer [P (3HB-co-5HV)] could be produced.
- This PHA synthase is suitable for biosynthesis of PHA that can be used in a variety of biomedical applications due to its ability to incorporate 4HB and 5HV lipase-degrading monomer sequences.
- this PHA synthase produces P (3HB-co-3HHx) and has properties similar to general purpose plastics such as polypropylene (PP) and low density polyethylene (LDPE), which is commercially useful PHA. It is evaluated as a copolymer.
- general purpose plastics such as polypropylene (PP) and low density polyethylene (LDPE), which is commercially useful PHA. It is evaluated as a copolymer.
- the present invention also describes PhaJ [PhaJ Ss (accession number: ALC30197)] capable of supplying an mcl monomer for PHA polymerization.
- PhaJ was discovered in Streptomyces strain CFMR7. It exhibits (R) -specific hydratase activity that supplies 3HHx-CoA from fatty acid ⁇ -oxidation to P (3HB-co-3HHx) during biosynthesis.
- the present invention discloses an isolated polynucleotide encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 having polymer synthase activity.
- SEQ ID NO: 1 is shown in FIG.
- the isolated polynucleotide is a polymer synthase gene.
- this polymer synthase gene is a polypeptide containing the amino acid sequence of SEQ ID NO: 1, or one or more amino acids are deleted from the amino acid sequence of SEQ ID NO: 1, replaced with the amino acid sequence of SEQ ID NO: 1, or in SEQ ID NO: 1.
- Polypeptides containing sequences added to the amino acid sequence can be encoded. Even if one or more amino acids in the sequence of SEQ ID NO: 1 are mutated such as deletion, substitution or addition, the polypeptide encoding the polypeptide containing the amino acid sequence is such that the polypeptide has polymer synthase activity.
- the gene of the present invention includes not only the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, but also its degenerate sequence encoding the same polypeptide except for the degenerate codon.
- the above mutations such as deletions, substitutions or additions, can be induced by known site-specific mutagenesis.
- an isolated polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 2 or a complementary sequence thereof is disclosed.
- SEQ ID NO: 2 is shown in FIG.
- Yet another embodiment of the invention is an isolated polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 in which T is replaced with U or a complementary sequence thereof.
- This polymer synthase gene is preferably cloned from the mangrove soil metagenomics.
- the polymer synthase gene is isolated from whole soil DNA obtained from mangrove soil.
- the gene of the present invention can be obtained by a polymerase chain reaction (PCR) amplification method using mangrove soil metagenomic DNA as a template, or a hybridization method using a DNA fragment having a nucleotide sequence thereof as a probe.
- PCR polymerase chain reaction
- PCR is used as a preferred method for obtaining a DNA fragment of a polymer synthase gene using whole mangrove soil metagenomic DNA as a template.
- metagenomic DNA is extracted from fresh mangrove soil samples. It is known in the art that the isolation of metagenomic DNA involves the use of commercially available kits such as the MoBio PowerSoil DNA isolation kit and the determination of total metagenomic DNA sequences using shotgun metagenomic sequencing. In order to obtain a DNA fragment containing a polymer synthase gene derived from soil metagenomics, it is preferable to prepare a probe.
- Well-conserved regions of the polymer synthase gene can be selected from known amino acid sequences and nucleotide sequences encoding them can be used to design oligonucleotides.
- primer pairs of amplified nucleotides are designed. The sequence of the primer pair is shown in FIG. 5, in which SEQ ID NO: 5 is used as the forward primer and SEQ ID NO: 6 is used as the reverse primer.
- the restriction site SwaI is placed in the polymer in the vector by designing a primer with a stop codon after the polymer synthase gene sequence and a SwaI restriction site in front of the ApaI restriction site. It was added after the synthase gene.
- primer pairs of amplified nucleotides are designed. The sequence of the primer pair is shown in FIG. 5, in which SEQ ID NO: 7 is used as the forward primer and SEQ ID NO: 8 is used as the reverse primer.
- the amplified DNA fragment can be digested with appropriate restriction enzymes such as HindIII and ApaI.
- the DNA fragment is then ligated into a suitable vector pre-cleaved with restriction enzymes that can be HindIII and ApaI.
- This vector is dephosphorylated by treatment with alkaline phosphatase prior to ligation.
- Yet another embodiment of the present invention is a recombinant vector containing an isolated polynucleotide.
- the isolated polypeptide is substituted, deleted, substituted or added with a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 having polymer synthase activity, or one or more amino acids.
- It is a recombinant vector encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1, which has polymer synthase activity.
- a plasmid capable of autonomous replication in a host microorganism is used as the vector.
- plasmids that can be applied as vectors include pBBR1MCS2 and pBBR1-I-GG18 (pBBR1MCS2 derivative cloning vector). These vectors are obtained by modifying commercially available vectors. Vectors that can autonomously replicate in two or more host cells and various shuttle vectors, such as Escherichia coli or Bacillus brevis, can also be used. Such vectors are also cleaved with restriction enzymes to obtain fragments thereof.
- the DNA fragment is ligated with the vector fragment using a conventional DNA ligase kit.
- the DNA fragment is annealed and ligated with the vector fragment to prepare a recombinant vector.
- Another invention discloses an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 (R) specific hydratase activity.
- SEQ ID NO: 3 is shown in FIG.
- the isolated polynucleotide is an enoyl-CoA hydratase gene.
- this enoyl-CoA hydratase gene is a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 3, or one or more amino acids are deleted from the amino acid sequence set forth in SEQ ID NO: 3, and the amino acid sequence set forth in SEQ ID NO: 3 is deleted. It is possible to encode a polypeptide containing a sequence substituted with or added to the amino acid sequence set forth in SEQ ID NO: 3.
- the polypeptide encoding the polypeptide containing the amino acid sequence is specific to the polypeptide (R). As long as it has hydratase activity, it is included in the gene of the present invention.
- a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 lacking methionine at position 1 is also included in the gene of the present invention.
- the gene of the invention includes not only the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, but also its degenerate sequence encoding the same polypeptide except for the degenerate codon.
- the above mutations, such as deletions, substitutions or additions can be induced by known site-specific mutagenesis.
- an isolated polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 4 or a complementary sequence thereof is disclosed.
- SEQ ID NO: 4 is shown in FIG.
- Yet another embodiment of the present invention is an isolated polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 in which T is replaced with U or a complementary sequence thereof.
- These polynucleotides encode polypeptides with (R) specific hydratase activity.
- This enoyl-CoA hydratase gene is preferably cloned from Streptomyces species CFMR7.
- the gene of the present invention can be obtained by a polymerase chain reaction (PCR) amplification technique using Streptomyces species CFMR7 genomic DNA as a template.
- PCR polymerase chain reaction
- primer pairs of amplified nucleotides are designed. The sequence of the primer pair is shown in FIG. 5, in which SEQ ID NO: 9 is used as the forward primer and SEQ ID NO: 10 is used as the reverse primer.
- the amplified DNA fragment can be digested with an appropriate restriction enzyme such as SwaI.
- the DNA fragment is then ligated into a suitable vector pre-cleaved with a restriction enzyme that can be SwaI.
- This vector is dephosphorylated by treatment with alkaline phosphatase prior to ligation.
- Yet another embodiment of the invention is a recombinant vector comprising an isolated polynucleotide, wherein the isolated polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 3 and is (R) specific.
- a plasmid capable of autonomous replication in a host microorganism is used as the vector.
- plasmids that can be applied as vectors include pBBR1MCS2 and pBBR1-I-GG18 (pBBR1MCS2 derivative cloning vector). These vectors are obtained by modifying commercially available vectors. Vectors capable of autonomously replicating in two or more host cells and various shuttle vectors, such as Escherichia coli or Bacillus brevis, can also be used. Such vectors are also cleaved with restriction enzymes to obtain fragments thereof.
- the DNA fragment is ligated with the vector fragment using a conventional DNA ligase kit.
- the DNA fragment is annealed and ligated with the vector fragment to prepare a recombinant vector.
- the transformant is obtained by introducing the recombinant vector into an appropriate host strain that is compatible with the expression vector constructed using the recombinant vector.
- the present invention is not limited to the use of a particular host strain as long as the target gene can be expressed in the recombinant vector.
- suitable microorganisms belong to the genera Cupriavidus, Bacillus, Pseudomonas (bacteria), Saccharomyces and Candida (yeast), COS and CHO cell lines (animal cells).
- the recombinant DNA of the present invention is a suitable promoter, a DNA fragment of the present invention, and a transcription termination sequence for ensuring autonomous replication in the host when a bacterium belonging to the genus Cupriavidus or Pseudomonas is used as a host strain. It is preferably constructed to consist of.
- the expression vector consists of, but is not limited to, pGEM-T and pBBR1MCS-2 derivatives.
- the promoter may be of any type as long as it can be expressed in the host. Examples of promoters derived from Cupriavidus necator, E. coli or phage include estimated C. coli. Included are the necator promoter, trp promoter, lac promoter, PL promoter, pR promoter and T7 promoter.
- Recombinant vectors can be introduced into host microorganisms using established methods.
- the host microorganism is Escherichia coli
- the calcium method and the electroporation method can be used.
- phage DNA when phage DNA is used, an in vitro packaging method can be adopted.
- an expression vector such as Yep13 or YCp50 is used. Therefore, a gal1 promoter or a gal10 promoter can be used as the promoter.
- Examples of the method for introducing the recombinant DNA into yeast include an electroporation method, a spheroplast method, and a lithium acetate method.
- expression vectors such as pcDNAI or pcDNAI / Amp are used. Therefore, as a method for introducing recombinant DNA into animal cells, an electroporation method or a potassium phosphate method can be used.
- the present invention comprises a step of culturing a transformant having the DNA according to any one of the above embodiments in a medium containing a polymerizable compound, and recovering a polymer formed and accumulated in the transformant.
- a process for producing a polymer including steps.
- the transformant medium is also used for microorganisms belonging to the genera Cupriavidus and Pseudomonas as hosts. These media contain carbon sources that can be assimilated with microorganisms with restricted nitrogen sources, inorganic salts or other organic nutrient sources, such as 0.01% to 0.1% by weight of the nutrient source. Contains a range of media.
- the carbon source is necessary for the growth of microorganisms, and at the same time, it is also the starting material for carbon such as glucose, fructose, sucrose or maltose. Further, as the carbon source, a fat-related substance having two or more carbon atoms can be used.
- Fats and oils include corn oil, soybean oil, benibana oil, sunflower oil, olive oil, coconut oil, palm oil, rapeseed oil, fish oil, whale oil, pig oil, bovine and other natural fats and oils; acetic acid, propionic acid, lauric acid, Fatty acids such as pentanoic acid, hexanic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linolenic acid, linoleic acid, myristic acid; ethanol, propanol, butanol, pentanol, hexanol, octanol, lauryl Examples thereof include alcohols such as alcohol, oleyl alcohol and palmitic acid, and esters thereof.
- inorganic substances include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, calcium chloride, magnesium sulfate and ammonium chloride.
- the culture is preferably carried out under aerobic conditions while shaking at 30 ° C. to 34 ° C. for 24 hours or longer, preferably 1 to 3 days.
- antibiotics such as ampicillin, kanamycin, gentamicin, antipyrine or tetracycline can be added to the culture.
- the polymer can be accumulated in the microorganism and the polymer can be recovered.
- the inducing substances such as isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) and indole acrylic acid (IAA) are added to the medium. You can also do it.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- IAA indole acrylic acid
- a medium such as RPMI-1640 or DMEM supplemented with fetal bovine serum can be used.
- culturing is usually carried out at 30 ° C. to 37 ° C. for 14 to 28 days in the presence of 5% CO2.
- antibiotics such as kanamycin or penicillin can be added to the medium.
- a polymer purification step can also be carried out.
- the transformant is recovered from the culture by centrifugation, washed with distilled water and hexane and dried. The dried transformant is then suspended in chloroform and heated to extract the polymer. The residue can be removed by filtration.
- methanol is added to this chloroform solution to precipitate the polymer. After removing the supernatant by filtration or centrifugation, the precipitate is dried to give a purified polymer. It is confirmed that the obtained polymer is desired by a usual method, for example, gas chromatography, nuclear magnetic resonance, or the like.
- This polymer synthase is a copolymer composed of a monomer unit 3-hydroxyalkonic acid represented by the formula (I) (where R represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). (Polymer) can be synthesized.
- the polymer is polyhydroxyalkanoate.
- the polymers are P (3HB-co-3HV random copolymer, [P (3HB-co-3HHx)] random copolymer, [P (3HB-co-3H4MV)] random copolymer, [P (3HB-co-4HB)] random. It can be a copolymer containing a copolymer and a [P (3HB-co-5HV)] random copolymer.
- a P (3HB-co-3HHx) copolymer can be produced in a high yield by using a polymer synthase of BPM-CPF-4. Since a large amount of a desired polymer can be obtained by using the above means, it can be used as a biodegradable material such as a thread or a film.
- the gene of the present invention can also be used for breeding of strains that highly produce P (3HB-co-3HHx) copolymers.
- the P (3HB-co-3HHx) copolymer is co-expressed with a polymer synthase (in this case, a polymer synthase of BPM-CPF-4) and enoyl-CoA hydratase to produce a higher 3HHx composition.
- a polymer synthase in this case, a polymer synthase of BPM-CPF-4
- enoyl-CoA hydratase to produce a higher 3HHx composition.
- the genes of the invention can be used to breed strains that produce P (3HB-co-3HHx) copolymers with higher 3HHx composition.
- P (3HB-co-4HB) and P (3HB-co-5HV) copolymers can be produced in high yield by using the polymer synthase of BPM-CPF-4. These copolymers are used in a variety of biomedical applications due to their ability to incorporate 4HB and 5HV lipase-degradable monomer sequences.
- Shotgun metagenome sequencing was performed using 125 bp pair-end sequencing using the Illumina HiSeq 2000 platform.
- Raw sequences (without filters) were submitted to the metagenomics RAST server (MG-RAST) for automatic sequence pretreatment (quality check) and gene annotation.
- Metagenomic data was deposited in the MG-RAS database under ID: mgm4512801.3 (Balik Pulau_mangrove).
- MG-RAST metagenomics RAST server
- RefSeq NCBI reference sequence
- a probe was prepared in order to obtain a DNA fragment containing a polymer synthase gene from metagenomic DNA.
- Two domain-specific oligonucleotides (SEQ ID NO: 5 and SEQ ID NO: 6) designed using the NCBI database as references were synthesized.
- the polymer synthase gene was amplified by PCR using these oligonucleotides as primers and the metagenomic DNA of mangrove soil as a template.
- the PCR conditions were as follows: 30 cycles at 94 ° C for 3 minutes; 94 ° C for 30 seconds, 57 ° C for 30 seconds and 72 ° C for 2 minutes; and a final step at 72 ° C for 10 minutes.
- the 1.7 kbp HindIII-ApaI nucleotide sequence from this fragment was determined by the Sanger method.
- a polymer synthase gene containing the nucleotide sequence of SEQ ID NO: 1 (1656) was obtained.
- Escherichia coli S17-1 was transformed by the calcium chloride method using a recombinant plasmid.
- the recombinant Escherichia coli thus obtained and C. coli.
- Necator PHB - 4 was transconjugated.
- Necator PHB - 4 was cultured overnight in 1.5 mL of LB medium and a nutrient-rich medium at 30 ° C., and the respective cultures (0.1 mL each) were combined and cultured on a shaker at room temperature for 1 hour. The mixture was then incubated for 30 minutes without shaking and then shaken again for 30 minutes. This microbial mixture was seeded on Simmons citrate agar containing 300 mg / L kanamycin and cultured at 30 ° C. for 2 days.
- necator PHB - 4 is made resistant to kanamycin by transferring the plasmid in recombinant E. coli into it, colonies grown on Simmons citrate agar are C. coli. It is a transformant of necator.
- Example 3 Synthesis of polymers with necator transformants
- a necator transformant in 50 mL of mineral medium (0.25 g / L magnesium sulfate heptahydrate, 3.32 g / L disodium hydrogen phosphate, 2.8 g / L phosphate) containing 1 ml / L of trace elements. It was inoculated with potassium dihydrogen (0.5 g / L ammonium chloride) and incubated in a flask at 30 ° C. 50 mg / L kanamycin was added to C.I. The microorganisms were cultured for 48 hours in addition to the medium for the necator transformant.
- potassium dihydrogen 0.5 g / L ammonium chloride
- necator transformant and PHB - 4 strains were inoculated into the above mineral medium supplemented with 10 g / L fructose and 6 g / L crude palm kernel oil (CPKO), respectively, and each strain was cultured in a 250 mL flask at 30 ° C. for 48 hours. did.
- a precursor carbon source was added for copolymer formation.
- 50 mg / L kanamycin was added to C.I. It was added to the medium of the necator transformant.
- the culture was collected by centrifugation, washed with distilled water and hexane (in the presence of CPKO), lyophilized, and weighed the dried microorganisms.
- 2 mL of a sulfuric acid / methanol mixture (15:85) and 2 mL of chloroform were added, the simple substance was sealed, and the mixture was heated at 100 ° C. for 140 minutes to decompose the polymer in the microorganism into methyl ester.
- Table 1 shows C.I. from fructose.
- the biosynthesis of PHA by necator transformants, a mixture of fructose with different precursor carbon sources and CPKO is shown.
- the reported values are mean ⁇ SD of 3 cultures.
- a PHA content in lyophilized cells 3HB: 3-hydroxybutyrate; 3HV: 3-hydroxyvalerate; 5HV: 5-hydroxyvalerate; 4HB: 4-hydroxybutyrate; 3H4MV: 3-hydroxy-4-methylvalerate 3HHx: 3-hydroxyhexanoate
- the transformant was able to utilize fructose for the production of P (3HB) homopolymers.
- Dry cell weight 3.7 ⁇ 0.1 g / L and polymer content: 75 ⁇ 9% by weight of microorganisms.
- Lower cell dry weights were obtained when CPKO was used as the sole source of carbon.
- the cell biomass of the transformant was 2.8 ⁇ 0.2 g / L, and the polymer content was 62 ⁇ 5% by weight of the microorganism.
- the accumulation of a P (3HB-co-3HHx) copolymer with 7 mol% 3HHx was observed in the transformant.
- the 3HV composition produced by the transformant was 13 ⁇ 1 mol%.
- the cellular biomass of the transformant was 2.5 ⁇ 0.1 g / L, and the polymer content produced by this transformant was 58 ⁇ 7% by weight of the microorganism.
- the 5HV composition produced by the transformant was 23 ⁇ 0 mol%.
- the cellular biomass of the transformant was 3.8 ⁇ 0.9 g / L, and the polymer content produced by this transformant was 67 ⁇ 8% by weight of the microorganism.
- the 4HB composition produced by the transformant was 14 ⁇ 1 mol%.
- the cellular biomass of the transformant was 2.8 ⁇ 0.1 g / L, and the polymer content produced by this transformant was 58 ⁇ 1% by weight of the microorganism.
- Isocaproic acid was added to fructose-supplemented cultures to investigate the formation of the P (3HB-co-3H4MV) copolymer.
- the 3HV composition produced by the transformant was 10 ⁇ 1 mol%.
- the cellular biomass of the transformant was 1.6 ⁇ 0.0 g / L, and the polymer content produced by this transformant was 45 ⁇ 5% by weight of the microorganism.
- the 3HHx composition produced by the transformant was 18 ⁇ 2 mol%.
- the cellular biomass of the transformant was 1.9 ⁇ 0.1 g / L, and the polymer content produced by this transformant was 44 ⁇ 1% by weight of the microorganism.
- the obtained polymer is confirmed by nuclear magnetic resonance.
- a total of 25 mg of polymer sample is dissolved in 1 mL of deuterated chloroform (CDCL3).
- PHA polymers were analyzed by 1 H NMR spectroscopy using Bruker AVANCE 500 (USA) operating at 500 MHz. The results are shown in FIGS. 6 and 7.
- Two specific oligonucleotides (SEQ ID NO: 9 and SEQ ID NO: 10) were synthesized to amplify the enoyl-CoA hydratase gene from the genomic DNA of Streptomyces species CFMR7.
- the enoyl-CoA hydratase gene was amplified by PCR.
- the PCR conditions were as follows: 30 cycles at 94 ° C for 3 minutes; 94 ° C for 30 seconds, 57 ° C for 30 seconds and 72 ° C for 2 minutes; and a final step at 72 ° C for 10 minutes.
- the nucleotide sequence of 850 bp SwaI-SwaI from this fragment was determined by the Sanger method.
- Escherichia coli S17-1 was transformed by the calcium chloride method using a recombinant plasmid.
- the recombinant Escherichia coli thus obtained and C. coli.
- Necator PHB - 4 was transconjugated.
- Necator PHB - 4 was cultured overnight in 1.5 mL of LB medium and a nutrient-rich medium at 30 ° C., and the respective cultures (0.1 mL each) were combined and cultured on a shaker at room temperature for 1 hour. The mixture was then incubated for 30 minutes without shaking and then shaken again for 30 minutes. This microbial mixture was seeded on Simmons citrate agar containing 300 mg / L kanamycin and cultured at 30 ° C. for 2 days.
- necator PHB - 4 is made resistant to kanamycin by transferring the plasmid in recombinant E. coli into it, colonies grown on Simmons citrate agar are C. coli. It is a transformant of necator.
- Example 6 Synthesis of polymers with necator transformants C.
- Each of the necator transformants was subjected to 50 mL of mineral medium (4.0 g / L NaH 2 PO 4 , 4.6 g / L Na 2 HPO 4 , 0.45 g / L K 2 SO 4 , 0.54 g / L Urea, 0.39 g / L sulfonyl 4 , 0.062 g / L CaCl 2 and 1 ml / L trace elements were inoculated and incubated in a flask at 30 ° C.
- the trace element solution was 15 g / dissolved in 0.1 M hydrochloric acid.
- L FeSO 4 ⁇ 7H 2 O consisted of 2.4g / L MnSO 4 ⁇ H 2 O, 2.4g / L ZnSO 4 ⁇ 7H 2 O and 0.48g / L CuSO 4 ⁇ 5H 2 O. 50 mg / L of canamycin was added to the medium for the C. necator transformant, and the microorganism was cultured for 48 hours.
- Each strain of the necator transformant was inoculated into the above mineral medium supplemented with 6 g / L of palm olein (PO) crude palm kernel oil (CPKO), and each strain was cultured in a 250 mL flask at 30 ° C. for 48 hours. 50 mg / L kanamycin was added to C.I. It was added to the medium of the necator transformant.
- PO palm olein
- CPKO crude palm kernel oil
- the culture was collected by centrifugation, washed with distilled water and hexane (in the presence of CPKO), lyophilized, and weighed the dried microorganisms.
- 2 mL of a sulfuric acid / methanol mixture (15:85) and 2 mL of chloroform were added, the simple substance was sealed, and the mixture was heated at 100 ° C. for 140 minutes to decompose the polymer in the microorganism into methyl ester. 1 mL of distilled water was added thereto, and the mixture was vigorously stirred.
- the obtained polymer is confirmed by nuclear magnetic resonance.
- a total of 25 mg of polymer sample is dissolved in 1 mL of deuterated chloroform (CDCL 3 ).
- PHA polymers were analyzed by 1 H NMR spectroscopy using Bruker AVANCE 500 (USA) operating at 500 MHz.
- a PHA content in lyophilized cells 3HB: 3-hydroxybutyrate; 3HHx: 3-hydroxyhexanoate
- the cell biomass of PHB - 4 / pBBR1MCS2_C BP-M-CPF4 was 4.9 ⁇ 0.5 g / L, and the polymer content was 53 ⁇ 6% by weight of the microorganism. 4 mol% of P (3HB-co-3HHx) copolymer having a 3HHx accumulation was observed, molecular weight of the copolymer (Mw) of 9.9 ⁇ 10 5 Da.
- the cell biomass of PHB - 4 / pBBR1MCS2_C BP-M-CPF4 was 5.6 ⁇ 0.3 g / L, and the polymer content was 58 ⁇ 2% by weight of the microorganism.
- PHB - 4 / pBBRMCS2_C cell biomass of BP-M-CPF4 _J Ss is 5.0 ⁇ 0.1g / L
- the polymer content was 54 ⁇ 4% by weight of the microorganism.
- Accumulation of 3HHx 12 mol% P (3HB-co-3HHx) copolymer was observed, and the molecular weight (Mw) of the copolymer was 8.0 ⁇ 10 5 Da.
- FIG. 8 shows 1 H NMR spectroscopy of a polymer produced using a phaJ-free transformant (PHB - 4 / pBBRMCS2_C BP-M-CPF4 ) and (a) PO or (b) CPKO as a carbon source. It is the analysis result by.
- the contents of the 3HHx monomer calculated by 1 H NMR spectroscopy were (a) 5 mol% and (b) 10 mol%, respectively.
- Figure 9 is a transformant containing a PhaJ - and (PHB 4 / pBBR1MCS2_C BP-M -CPF4_ J Ss), 1 H NMR spectroscopy (a) PO or (b) polymers produced using CPKO as a carbon source This is the result of analysis by law.
- the contents of 3HHx monomers calculated by 1 H NMR spectroscopy were (a) 13 mol% and (b) 20 mol%, respectively.
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Abstract
Description
なお、本明細書に記載の実施形態は、本発明の範囲を限定するものではなく、当業者であれば、本発明が目的を実行し、上述した目的及び利点、並びにそれらに固有の目的及び利点を得るためによく適合していることを容易に理解するであろう。
マングローブ土壌メタゲノムからのポリマー合成酵素遺伝子のクローニング
最初に、メタゲノムDNAをMoBio PowerSoil DNA単離キットを用いて直接マングローブ土壌から単離した。
C.necator形質転換体の調製
HindIII-ApaIポリマー合成酵素遺伝子断片を、先に同じ制限酵素で切断したクローニングベクターpCR(登録商標)4-TOPO(登録商標)(米国、Invitrogen)に最初に挿入した。次いで、断片をHindIII及びApaI制限酵素で再び消化し、得られたHindIII-ApaIポリマー合成酵素遺伝子断片を、Cupriavidus属に属する微生物において発現可能な組換えベクターpBBR1MCS-2に挿入し、得られた組換えプラスミドをコンジュゲーション転写法によりCuprividus necator PHB-4(DSM541)(ポリマーの合成能力が欠損した株)に形質転換した。
C.necator形質転換体によるポリマーの合成
C.necator形質転換体を、1ml/Lの微量元素を含有する50mLのミネラル培地(0.25g/L硫酸マグネシウム七水和物、3.32g/Lリン酸水素二ナトリウム、2.8g/Lリン酸二水素カリウム、0.5g/L塩化アンモニウム)に接種し、フラスコ内にて30℃でインキュベートした。50mg/LのカナマイシンをC.necator形質転換体用の培地に加え、微生物を48時間培養した。
出発培養物からの3%接種物(O.D=~4.5)を250mL三角フラスコ中の50mLのMMに移し、30℃、48時間、200rpmでインキュベートした。報告された値は、3回培養の平均値±SDである。
a凍結乾燥細胞におけるPHA含量
3HB:3-ヒドロキシブチラート;3HV:3-ヒドロキシバレレート;5HV:5-ヒドロキシバレレート;4HB:4-ヒドロキシブチラート;3H4MV:3-ヒドロキシ-4-メチルバレレート;3HHx:3-ヒドロキシヘキサノエート
Streptomyces種CFMR7由来のEnoyl-CoAヒドラターゼ遺伝子のクローニング
最初に、ゲノムDNAを単離した。Streptomyces種CFMR7 QIAamp DNAミニキット。
C.necator形質転換体の調製
SwaI-SwaIエノイル-CoAヒドラターゼ遺伝子断片を、以前に構築した([0099])BP-M-CPF4のポリマー合成酵素を有する組換えベクターpBBR1MCS-2に挿入し、得られた組換えプラスミドをコンジュゲーション転写法によりCuprividus necator PHB-4(DSM541)(ポリマーの合成能力が欠損した株)に形質転換した。[0099]の組換えベクター構築物も、エノイル-CoAヒドラターゼの効果を比較するための陰性対照として用いた。
C.necator形質転換体によるポリマーの合成
C.necator形質転換体の各々を、50mLのミネラル培地(4.0g/L NaH2PO4、4.6g/L Na2HPO4、0.45g/L K2SO4、0.54g/L Urea、0.39g/L MgSO4、0.062g/L CaCl2及び1ml/Lの微量元素に接種し、フラスコ内にて30℃でインキュベートした。微量元素溶液は、0.1M塩酸に溶解した15g/L FeSO4・7H2O、2.4g/L MnSO4・H2O、2.4g/L ZnSO4・7H2O及び0.48g/L CuSO4・5H2Oからなるものであった。50mg/LのカナマイシンをC.necator形質転換体用の培地に加え、微生物を48時間培養した。
出発培養物からの3%接種物(O.D=~4.5)を250mL三角フラスコ中の50mLのMMに移し、30℃、48時間、200rpmでインキュベートした。表中にGC及びGPCによる分析により得られた結果を示した。この数値は、3回培養の平均値±SDである。
a凍結乾燥細胞におけるPHA含量
3HB:3-ヒドロキシブチラート;3HHx:3-ヒドロキシヘキサノエート
Claims (20)
- 配列番号1に記載のアミノ酸配列、又は、1つ以上のアミノ酸が置換、欠失若しくは付加された配列番号1に記載のアミノ酸配列を含み、かつポリマー合成酵素活性を有するポリペプチドをコードする単離されたポリヌクレオチド。
- 配列番号2に記載のヌクレオチド配列、若しくは、1つ以上のヌクレオチドが置換された配列番号2に記載のヌクレオチド配列、又はその相補配列を含む、請求項1に記載の単離されたポリヌクレオチド。
- 配列番号2に記載のヌクレオチド配列において、チミンがウラシルによって置換されているヌクレオチド配列又はその相補配列を含む、請求項1に記載の単離されたポリヌクレオチド。
- 請求項1~3のいずれか1項に記載の単離されたポリヌクレオチドを含む組換えベクター。
- プラスミド又はファージである、請求項4に記載の組換えベクター。
- 請求項4に記載の組換えベクターによって形質転換された形質転換体。
- 重合性材料を含む培地で、請求項6に記載の形質転換体を培養する工程と、
前記培養された培地からポリマーを回収する工程と、
を含むポリマーの製造方法。 - 請求項1~3のいずれか1項に記載の単離されたポリヌクレオチドをゲノムに有する組換え株。
- 重合性材料を含む培地で、請求項8に記載の組換え株を培養する工程と、
前記培養された培地からポリマーを回収する工程と、
を含むポリマーの製造方法。 - 前記ポリマーがポリヒドロキシアルカノエートである、請求項7又は9に記載のポリマーの製造方法。
- 配列番号3に記載のアミノ酸配列、又は、1つ以上のアミノ酸が置換、欠失若しくは付加された配列番号1に記載のアミノ酸配列を含み、かつヒドラターゼ活性を有するポリペプチドをコードする単離されたポリヌクレオチド。
- 配列番号4に記載のヌクレオチド配列、若しくは、1つ以上のヌクレオチドが置換された配列番号2に記載のヌクレオチド配列、又はその相補配列を含む、請求項11に記載の単離されたポリヌクレオチド。
- 配列番号4に記載のヌクレオチド配列において、チミンがウラシルによって置換されているヌクレオチド配列、又はその相補配列を含む、請求項11に記載の単離されたポリヌクレオチド。
- 請求項11~13のいずれか1項に記載の単離されたポリヌクレオチドを含み、及び、ポリマー合成酵素活性を有するポリペプチドをコードするポリヌクレオチドを更に含む組換えベクター。
- プラスミド又はファージである、請求項14に記載の組換えベクター。
- 請求項14に記載の組換えベクターによって形質転換された形質転換体。
- 重合性材料を含む培地で、請求項14に記載の形質転換体を培養する工程と、
前記培養された培地からポリマーを回収する工程と、
を含むポリマーの製造方法。 - 請求項11~13のいずれか1項に記載の単離されたポリヌクレオチドをゲノムに有する組換え株。
- 重合性材料を含む培地で、請求項18に記載の組換え株を培養する工程と、
前記培養された培地からポリマーを回収する工程と、
を含むポリマーの製造方法。 - 前記ポリマーがポリヒドロキシアルカノエートである、請求項17又は19に記載のポリマーの製造方法。
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