WO2015115619A1 - R体特異的エノイル-CoAヒドラターゼ遺伝子の発現が調節された微生物及びそれを用いたポリヒドロキシアルカノエート共重合体の製造方法 - Google Patents
R体特異的エノイル-CoAヒドラターゼ遺伝子の発現が調節された微生物及びそれを用いたポリヒドロキシアルカノエート共重合体の製造方法 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01017—Enoyl-CoA hydratase (4.2.1.17), i.e. crotonase
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- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01074—Long-chain-enoyl-CoA hydratase (4.2.1.74)
Definitions
- the present invention relates to a recombinant microorganism in which the expression of an R-form-specific enoyl-CoA hydratase gene is regulated, and a method for producing a polyhydroxyalkanoate copolymer having a controlled monomer composition ratio using the microorganism.
- PHA Polyhydroxyalkanoate
- PHA is a polyester-type organic molecular polymer produced by a wide range of microorganisms.
- PHA is a biodegradable thermoplastic polymer, and PHA can also be produced from renewable resources. For these reasons, attempts have been made to industrially produce PHA as an environmentally conscious material or a biocompatible material and use it in various industries.
- PHA poly-3-hydroxybutyric acid
- 3HB 3-hydroxybutyric acid
- P (3HB) is a thermoplastic polymer and is biologically decomposed in the natural environment, and thus has attracted attention as an environmentally friendly plastic.
- P (3HB) is hard and brittle because of its high crystallinity, and its application range is limited in practical use. In order to expand the application range, it was necessary to give flexibility to P (3HB).
- P (3HB-co-3HV) a PHA copolymer composed of 3HB and 3-hydroxyvaleric acid (hereinafter referred to as 3HV) and a production method thereof have been developed (Patent Document 1, Patent Document 2). Since P (3HB-co-3HV) is more flexible than P (3HB), it was considered to be applicable to a wide range of applications. However, in practice, even if the 3HV mole fraction in P (3HB-co-3HV) is increased, the accompanying change in physical properties is scarce, which is particularly required for processing into films, sheets, flexible packaging containers, etc. Since flexibility does not improve as much, it is used only in limited fields such as shampoo bottles and disposable razor handles.
- P (3HB-co-3HH) a PHA copolymer comprising 3HB and 3-hydroxyhexanoic acid (hereinafter sometimes referred to as 3HH) (hereinafter sometimes referred to as P (3HB-co-3HH)).
- 3HH 3-hydroxyhexanoic acid
- Patent Document 4 Patent Document 4
- P (3HB-co-3HH) is fermentatively produced using a wild strain of Aeromonas caviae isolated from soil and using fatty acids such as oleic acid and palmitic acid as a carbon source
- the 3HH composition ratio of the obtained P (3HB-co-3HH) was 15 mol% when oleic acid was used as the carbon source and 5 mol% when palmitic acid was used as the carbon source.
- Non-patent Document 1 Research on the physical properties of P (3HB-co-3HH) has also been conducted (Non-patent Document 1). In this report, fatty acids having 12 or more carbon atoms are used as the sole carbon source. Caviae is cultured, and P (3HB-co-3HH) having a 3HH composition ratio of 11 to 19 mol% is produced by fermentation. As P (3HB-co-3HH) increases in composition ratio from 3HH, it becomes more flexible from hard and brittle properties such as P (3HB), and P (3HB- co-3HV) was shown to exhibit greater flexibility. That is, P (3HB-co-3HH) can have a wide range of physical properties applicable from hard polymers to soft polymers by changing the 3HH composition ratio. Therefore, P (3HB-co-3HH) has a low 3HH composition ratio. ) Is required for hardness, such as the housing of a television, while P (3HB-co-3HH) with a high 3HH composition ratio is required for flexibility, such as a film. It can be used in a wide range of
- transformation with PHA synthase expression plasmids such as pJRDDE32 and pJRDEE32d13, in which polyester synthase gene, R-body-specific enoyl CoA hydratase gene and the like are introduced into pJRD215 (ATCC 37533), using Capriavidas necatol (C. necator) as a host.
- the PHA productivity of the body has been investigated (Patent Document 5, Non-Patent Document 2).
- the cell mass after cultivation of the strain was originally as low as 4 g / L, but the cell mass was 45 g / L, the polymer content was 62.5%, 3HH by improving the culture conditions of the strain using vegetable oil as a carbon source.
- the R-form-specific enoyl CoA hydratase gene was transferred to C.I. It has also been reported that the 3HH composition ratio has been improved by incorporating it into the chromosomal DNA of necator (Patent Document 7, Non-Patent Document 3). In this report, C.I. The 3HH composition ratio is increased to 10.5 mol% by inserting a plurality of R-form-specific enoyl CoA hydratase genes into the pha operon region containing the nephar pha synthase gene.
- the present invention relates to breeding of a microorganism producing a polyhydroxyalkanoate (PHA) copolymer having a controlled monomer composition ratio, and polyhydroxyalkanoate (PHA) copolymer having a controlled monomer composition ratio using the microorganism.
- the purpose is to ferment the union.
- the present inventors have controlled the 3HH monomer synthesis pathway by regulating the expression of the R-form-specific enoyl CoA hydratase gene endogenous to the microorganism, The inventors have found that the monomer composition ratio of the accumulated PHA copolymer can be efficiently controlled, and have completed the present invention.
- the present invention includes the following features.
- microorganism according to any one of (1) to (4), further comprising a polyhydroxyalkanoate synthase gene.
- (11) One or more selected from the group consisting of an E. coli-derived promoter, a phaC1 gene promoter, and a phaC1 gene modified promoter, wherein the base sequence upstream of the R-form-specific enoyl CoA hydratase gene is introduced by substitution or insertion
- microorganism according to any one of (2) to (11), further comprising an SD sequence downstream of the promoter sequence.
- An expression control DNA comprising any one of the promoters consisting of the base sequences represented by SEQ ID NOs: 47 to 50 and the base sequence represented by any one of SEQ ID NOs: 51, 52 and 60 to 66.
- polyhydroxyalkanoate copolymer is a copolymer containing 3-hydroxyhexanoic acid (3HH) monomer as a structural unit.
- a PHA copolymer having an industrially useful desired monomer composition ratio can be produced by fermentation.
- a microorganism having an R-form-specific enoyl CoA hydratase gene on genomic DNA and the expression of the gene being regulated is a microorganism having an R-form-specific enoyl CoA hydratase gene on genomic DNA,
- the base sequence upstream of the R-form-specific enoyl CoA hydratase gene includes a modification consisting of substitution, deletion, insertion and / or addition of one or more bases, whereby the R-form-specific enoyl CoA hydratase gene
- Provided is a microorganism characterized in that expression is regulated.
- genomic DNA includes not only chromosomal DNA (also simply referred to as “chromosome”) but also, for example, megaplasmid DNA (also simply referred to as “megaplasmid”). It is a chromosome.
- the “R-form-specific enoyl CoA hydratase gene” used in the present specification is converted from enoyl CoA, which is an intermediate of fatty acid ⁇ -oxidation system, to (R) -3-hydroxyacyl CoA, which is a source of PHA monomer. It is a gene that encodes an R-form specific enoyl CoA hydratase (PhaJ), which is an enzyme.
- the microorganism used in the present invention is not particularly limited as long as it has a R-form-specific enoyl CoA hydratase gene on the genomic DNA, and a wild-type strain originally having an R-form-specific enoyl CoA hydratase gene, or an artificial strain of the wild-type strain is used.
- the mutant may be a mutant obtained by performing a mutation treatment, or a recombinant strain in which an exogenous R-form-specific enoyl CoA hydratase gene is inserted or replaced on genomic DNA by genetic engineering techniques.
- microorganisms include bacteria, yeasts, filamentous fungi, and the like, preferably bacteria, such as the genus Ralstonia, the genus Capriavidus, the genus Watertersia, and Aeromonas.
- bacteria such as the genus Ralstonia, the genus Capriavidus, the genus Watertersia, and Aeromonas.
- Preferred examples include bacteria of the genus (Aeromonas), the genus Escherichia, the genus Alcaligenes, and the genus Pseudomonas.
- a bacterium belonging to the genus Ralstonia, Capriavidas, Aeromonas, or Wautercia more preferably a bacterium belonging to the genus Ralstonia or Aeromonas, especially Preferably, it is Capriavidus necatol or Aeromonas hydrophila.
- Bacteria-derived R-form-specific enoyl CoA hydratase is available in public databases such as GenBank of NCBI (USA).
- YP — 72579.1, YP — 728561.1, YP — 728829.1, etc. are registered and known as R-form specific enoyl CoA hydratase derived from necator.
- the microorganism includes a modification in which the base sequence upstream of the R-form-specific enoyl CoA hydratase gene on genomic DNA comprises substitution, deletion, insertion and / or addition of one or more bases.
- the expression of the R-form specific enoyl CoA hydratase gene is regulated.
- the substitution, deletion, insertion and / or addition increases the intensity of expression of the R-form specific enoyl CoA hydratase gene to (positive (+) or negative ( ⁇ ), preferably compared to the wild type. There is no particular limitation as long as it can be changed to positive (+).
- Upstream of the R-form specific enoyl CoA hydratase gene may contain one or more base modifications in the promoter sequence, the SD sequence, or both sequences that regulate the expression of the gene, or Base sequences other than promoter sequences and SD sequences may also contain one or more base modifications.
- an R-form-specific enoyl-CoA hydratase gene upstream ie, wild-type
- SD sequence or other upstream of the gene within 10,000 bases on the genomic DNA of the microorganism.
- Modification ie, substitution, deletion, insertion and / or addition, preferably substitution or insertion of one or more bases
- is introduced into the base sequence preferably into the promoter sequence, the SD sequence, or both.
- the intensity of expression of the R-form-specific enoyl CoA hydratase gene may be changed, or the R-form-specific enoyl CoA hydratase may be added to the base sequence upstream of the R-form-specific enoyl CoA hydratase gene on the genomic DNA. Insert DNA that can regulate gene expression, or place it with any upstream sequence. Introduced by, it may control the intensity of expression of the R-specific enoyl CoA hydratase genes.
- the nucleotide sequence of that site A part of the expression control DNA can be substituted with a promoter sequence and / or an SD sequence, or the expression control DNA can be inserted into the site.
- the introduced (in other words, exogenous) expression control DNA is, for example, the promoter sequence, SD sequence, or modified sequence thereof, or the homologous or heterologous R-specific enoyl CoA hydratase gene derived from the homologous or heterologous microorganism.
- the promoter of the R-form specific enoyl CoA hydratase gene derived from the same type of microorganism a promoter sequence of a different R-type specific enoyl CoA hydratase gene in the same type of microorganism may be used, or the same in the same type of microorganism.
- the promoter sequence of the R-form specific enoyl CoA hydratase gene may be introduced at a different position. The same applies to SD sequences derived from homologous microorganisms.
- one or more bases refers to an expression regulatory region (including a promoter region and / or an SD sequence region, upstream of the translation start site of the R-form specific enoyl CoA hydratase gene,
- the promoter region may also include an operator sequence.
- substitution, deletion, insertion and / or addition of one or more bases is the introduction of one or more base mutations that enable the expression of the gene to be regulated.
- “several” in the present specification refers to an integer of 2 to 10, preferably an integer of 2 to 7.
- regulatory expression or “make expression regulatable” refer to mRNA synthesis and subsequent protein synthesis by transcription of an R-form specific enoyl CoA hydratase gene. It means that control is preferably performed so that the level is substantially equal to or higher than that of the case, or less, or less.
- the sacB gene derived from Bacillus subtilis coexists, and the gene is obtained by homologous recombination at the second stage.
- Method for easily isolating a microbial strain from which sucrose has dropped as a sucrose-added medium resistant strain also can be utilized, the method is not particularly limited as long insert / replace any DNA on the genome DNA.
- a “promoter” is a regulatory sequence involved in the efficiency of the transcription initiation reaction of a gene, and includes both a regulatory sequence that works relatively close to the transcription start point and a regulatory sequence that works away from the transcription start point. It has a broad meaning to include.
- the promoter usually contains sequences from the ⁇ 35 region to the ⁇ 10 region upstream of the transcription start point (+1) of the gene, but the sequence of these regions is often depending on the prokaryotic species.
- its consensus sequence in E. coli is known to be, for example, TTGACA (SEQ ID NO: 53) and TATAAT (SEQ ID NO: 54).
- the SD sequence is generally a sequence that is located several bases to several tens of bases upstream of the translation start point (+1) of the gene, and is complementary or partially complementary to the 3 ′ end portion of 16S rRNA.
- the ribosome binds to the mRNA, and translation is started from the translation initiation codon immediately downstream thereof. Therefore, the SD sequence is located between the transcription start point and the translation start codon.
- the untranslated region upstream of the start codon in mRNA containing the SD sequence is generally called 5'UTR and may affect the stability of the mRNA.
- the SD sequence is located between the transcription start point and the translation start point of the structural gene, and encodes the ribosome binding sequence on mRNA involved in the translation of the structural gene (protein synthesis), and the ribosome binding sequence.
- the position where the above expression regulatory region is altered is not particularly limited as long as the expression can be regulated upstream in the genomic DNA region where the target R-form specific enoyl CoA hydratase gene is present. Is preferably substituted or inserted at a position closer to the R-form-specific enoyl CoA hydratase gene.
- substitution or insertion may be performed within 10,000 bases upstream of the R-form-specific enoyl CoA hydratase gene, preferably substitution or insertion within 1000 bases upstream, substitution within 500 bases upstream Alternatively, it is more preferable to insert, and it is particularly preferable to substitute or insert within 50 bases upstream.
- the R-form-specific enoyl CoA hydratase gene may form an operon with other genes such as other metabolic genes and enzymes involved in PHA synthesis.
- other genes such as other metabolic genes and enzymes involved in PHA synthesis.
- it is upstream of the R-form specific enoyl CoA hydratase gene.
- an expression regulatory sequence having a promoter and an SD sequence introduced from the outside by substitution or insertion is included between the other structural gene present and the R-form-specific enoyl CoA hydratase gene.
- the above C.I. Depending on the type of microorganism such as necator, there may be a plurality of R-form-specific enoyl CoA hydratase genes.
- the R-form-specific enoyl CoA hydratase gene that is subject to expression regulation is not particularly limited, and any one of a plurality of R-form-specific enoyl CoA hydratase genes may be targeted, or 2 More than one may be targeted.
- Necator originally has a chromosome and a megaplasmid as genomic DNA, and the R body-specific enoyl CoA hydratase gene that is subject to regulation of expression in the present invention may be present in any of them. It is preferably but present on the chromosome.
- the promoter and SD sequence contained in the expression regulatory sequence to be modified (or mutated) such as substitution and insertion upstream of the R-form-specific enoyl CoA hydratase gene are not particularly limited, and R-form-specific enoyl CoA Any sequence that can regulate the expression of the hydratase gene can be used.
- these promoter and SD sequence may be the same or different from the promoter and / or SD sequence originally possessed by the target microorganism, or a microorganism species different from the target microorganism (for example, Promoters and / or SD sequences derived from different genera, species or strains), or modified promoters and / or modified SD sequences obtained by modifying these sequences.
- the promoter contained in the expression control DNA is not particularly limited. substitution of at least one base in the phaC1 promoter (REP) which is the promoter of the PHA synthase gene (phaC1 gene) possessed by necator, the -35 region and / or the -10 region of the phaC1 promoter (REP), and the sequence between them
- REP phaC1 promoter
- modified REP modified phaC1 promoter obtained by changing by deletion, insertion and / or addition
- coli-derived promoter for example, trc promoter represented by SEQ ID NO: 47, SEQ ID NO: 49 lac promoter, lacUV5 promoter represented by SEQ ID NO: 50, trp promoter represented by SEQ ID NO: 48, and the like.
- E. coli-derived promoters such as trc promoter, lacUV5 promoter, trp promoter and the like are preferable in that they have a high effect of enhancing the expression of R-form-specific enoyl CoA hydratase gene.
- the modified REP include a promoter including the base sequence represented by any of SEQ ID NOs: 55 to 59, and a promoter including the base sequence represented by SEQ ID NO: 59 is preferable.
- the modified REP is C.I. It is obtained by modifying the sequence of the phaC1 promoter (REP), which is the promoter of the phaC1 gene, which is the PHA synthase gene of necator.
- the phaC1 promoter (REP) is also called a pha operon promoter because the phaC1 gene is present on the genomic DNA in the form of an operon with the phaA gene and the phaB1 gene.
- the phaC1 promoter (REP) to be modified is preferably C.I.
- Necator has a base sequence represented by SEQ ID NO: 67 (TTGACAGCCGCGTGCGTTGCAAGGCAACAAAT) derived from H16 strain.
- the ⁇ 35 region and the ⁇ 10 region are each 6 bases from the first base (for example, TTGACA (the same sequence as SEQ ID NO: 53)) and 6 bases from the 25th base (for example, AACAT (SEQ ID NO: 68)).
- the modified REP is the above-mentioned C.I. having substitution, addition, insertion and / or deletion, preferably substitution, of one or several bases, preferably 1 to 4 bases, in the ⁇ 35 region and ⁇ 10 region of the phaC1 promoter derived from necator Also obtained by changing the distance between the -35 region and the -10 region by deletion or addition of one or several bases, preferably 1-2 bases, and has promoter activity .
- “several” means an integer of 2 to 10, for example, an integer of 2 to 7, an integer of 2 to 5, an integer of 2 to 4, an integer of 2 to 3, and the like.
- the term “having promoter activity” refers to having an ability to bind RNA promoter to a promoter and initiate transcription (mRNA synthesis).
- the modified REP is a promoter having a base sequence represented by any of SEQ ID NOs: 55 to 59, for example.
- the nucleotide sequences of SEQ ID NOs: 55 to 59 are C.I. Since this is a modified phaC1 promoter sequence of necator H16 strain, if the strain is different, the modified REP may be different from the base sequence of SEQ ID NOs: 55 to 59, one or several in the wild type promoter sequence of the strain, Preferably, it consists of a sequence having 1 to 4 base substitutions, additions, insertions and / or deletions.
- the modified REP has a change in promoter activity or transcription efficiency compared to the wild-type phaC1 promoter (REP) by substitution, addition, insertion and / or deletion of the above base, and is useful for controlling the expression intensity of the structural gene. It is.
- the change in the promoter activity means that the activity is increased compared to the wild-type phaC1 promoter (REP), the activity is equivalent or reduced, for example, within 80%, or the wild-type phaC1 promoter is used. Any gene expression level that is higher than or equal to the gene expression level when obtained, or that can be obtained within 80% is obtained, and can be appropriately selected depending on the purpose.
- the modification of the phaC1 promoter can be performed, for example, by the following method.
- Synthesis of a nucleic acid having a substitution, addition, insertion and / or deletion of one or several, preferably 1 to 4 bases in the phaC1 promoter (REP) sequence is carried out by mutagenesis by PCR, site-directed mutagenesis. Or a known method such as automatic nucleic acid synthesis. Briefly describing a commonly used mutagenesis method by PCR, this method can be performed by combining, for example, a mutagenesis primer and a PCR method.
- the primer for mutagenesis is designed by designing an oligonucleotide capable of specifically amplifying the target nucleic acid based on a known method, and introducing the desired base mutation (substitution, addition, insertion and / or deletion). Can be produced.
- any conditions may be used as long as a nucleic acid containing a modified promoter can be amplified.
- a plasmid into which a double-stranded DNA having a size of about 30 bp to 900 bp or more (for example, about 30 bp to 1200 bp) including a promoter sequence derived from the wild-type phaC1 gene is constructed is constructed, and the base to be amplified This includes annealing the sense and antisense mutagenesis primers so as to sandwich the sequence, and performing an amplification reaction of 20 to 40 cycles.
- the SD sequence contained in the expression regulatory sequence is not particularly limited.
- the SD sequence of the phaC1 gene represented by SEQ ID NO: 51 (phaC1SD sequence)
- at least one base of the phaC1SD sequence is replaced.
- SEQ ID NO: 52 (TCCTCTCT), SEQ ID NO: 60 (TGGTGAGA), SEQ ID NO: 61 (ATATAGA), SEQ ID NO: 62 (AGTGAGA), SEQ ID NO: 63 (TGGTGTGA), SEQ ID NO: 64 (TGGAGTGA)
- a modified SD sequence represented by SEQ ID NO: 65 (AGATAGA) and SEQ ID NO: 66 (AGATAGA) and the like, and a modified SD sequence represented by SEQ ID NO: 52 is preferred.
- the modified SD sequence is, for example, A. It can be obtained by modifying the phaC1SD sequence predicted by Steinbuchel et al. (J. Bacterial. 1991, 173 (1): 168).
- the phaC1SD sequence to be modified in this case is C.I. It is located about 17 bases upstream from the translation start point of the phaC1 gene on the genomic DNA of necator, and is specifically represented by the base sequence of AGAGAGA (SEQ ID NO: 69).
- the modified SD sequence is a “modified phaC1SD sequence” obtained by substituting one or several, preferably 1 to 7 bases of the SD sequence of the phaC1 gene.
- a specific example of the modified phaC1SD sequence is DNA containing the base sequence represented by any of SEQ ID NOs: 52 and 60 to 66 described above.
- the modified phaC1SD sequence has a translational activity and / or stability of mRNA that is changed by the substitution of the base as compared with the case of using the wild-type phaC1SD sequence, and is useful for controlling the expression intensity of the structural gene.
- the translational activity and / or the change in mRNA stability is an increase compared to the wild-type phaC1SD sequence, an equivalent one, or a decrease within, for example, 60%, 40%, or 25%.
- a gene expression level (or protein production amount) that is higher than, or equivalent to, for example, within 60%, within 40%, or within 25% compared to when the wild type phaC1SD sequence is used. Any of these can be selected as appropriate according to the purpose.
- the modified phaC1SD sequence that is, the synthesis of a nucleic acid into which a mutation such as a base substitution has been introduced into the phaC1SD sequence is the same as described above.
- Mutation introduction method by PCR, site-directed mutagenesis method, automatic nucleic acid synthesis method, etc. can carry out by the well-known method. For example, it can be performed by combining a primer for mutagenesis and a PCR method.
- the primer for mutagenesis is designed based on a known method to design an oligonucleotide capable of specifically amplifying the target gene fragment, and the desired base mutation (substitution, addition, insertion and / or deletion) What is necessary is just to introduce and produce.
- As the amplification conditions by PCR any conditions may be used as long as the target nucleic acid can be amplified.
- the SD sequence is located between the transcription start point and the translation start point as an expression regulator, and is located several bases to a dozen bases upstream from the translation start point. Further, the untranslated region upstream of the start codon including the SD sequence is generally called 5'UTR and may affect the stability of mRNA. Therefore, introduction of mutations into the SD sequence and the 5 'UTR containing it may change the stability of mRNA, resulting in a change in gene expression intensity.
- the SD sequence is located between the transcription start point and the translation start point of the structural gene, and the ribosome binding sequence on mRNA involved in the translation of the structural gene (protein synthesis), and the ribosome binding sequence. Refers to the coding DNA sequence.
- the expression control DNA of the present invention those containing a promoter and an SD sequence as exemplified above are preferable.
- Such expression-regulating DNA has a promoter and an SD sequence that are arranged at appropriate positions where they can respectively exhibit a promoter function and a ribosome binding function, and can be used for gene expression.
- the above modified REP is contained together with the known SD sequence, or the expression containing the modified SD sequence together with the known promoter. Regulatory DNA can also be made and used.
- the expression control DNA of the present invention may contain appropriate DNA related to expression control, for example, within 900 bases, 500 bases, 300 bases or 200 bases upstream of the phaC1 gene.
- It may contain a base sequence within bases or a base sequence within 900 bases, within 500 bases, within 300 bases or within 200 bases upstream of the structural gene to be regulated. Further, the size of such a base sequence may exceed 900 bases as long as it is involved in expression regulation, but is preferably within 900 bases.
- the placement of the above-described expression control DNA by inserting a promoter and an SD sequence can be performed based on a known method. For example, it is achieved by amplifying a fragment containing each sequence by the PCR method, cleaving the obtained amplified fragment with an appropriate restriction enzyme, and binding to an appropriate position.
- the expression control DNA is not particularly limited as long as the expression can be regulated upstream of the structural gene targeted for expression regulation, but is bound to a position closer to the structural gene of interest. It is preferable.
- the target structural gene when it is on the genomic DNA, it may be joined by substitution or insertion within 10,000 bases upstream of the genomic DNA region, or by substitution or insertion within 1000 bases upstream It is preferable to be bonded, and it is more preferable to be bonded by substitution or insertion within 500 bases upstream, and it is particularly preferable to be bonded by replacement or insertion within 50 bases upstream.
- the target structural gene forms an operon with another structural gene, such as an R-form-specific enoyl CoA hydratase gene such as necator
- the target is affected without affecting the expression of the other structural gene.
- an expression regulatory sequence is inserted or replaced between another structural gene existing upstream from the target structural gene and the target structural gene. It is preferred that they are bonded.
- an expression regulatory sequence is bound to the original promoter region in the operon or its periphery by substitution or insertion.
- the promoter in the expression control sequence is the target structural gene.
- the target structural gene is not particularly limited upstream as long as its expression can be regulated, it is preferable that it is bound at a position relatively close to the target structural gene.
- it may be combined by substitution or insertion within 10,000 bases upstream, preferably by substitution or insertion within 1000 bases upstream, and preferably substituted or inserted within 500 bases upstream. Is more preferable, and it is particularly preferable that binding is performed by substitution or insertion within 100 bases upstream.
- the SD sequence since the SD sequence is involved in the translation activity from mRNA to protein, it must be located between the transcription start point downstream of the promoter and the translation start point. It is preferable to place it upstream from the base to a dozen bases.
- the expression control DNA is, for example, an SD sequence of the phaC1 gene represented by SEQ ID NO: 51, or SEQ ID NOs: 52, 60 to 66 at an appropriate position downstream of any one of the promoter sequences represented by SEQ ID NOs: 47 to 50. More preferably, the modified SD sequence represented by any one of the nucleotide sequences is present.
- the expression control DNA can contain any DNA sequence other than the promoter region and the SD region, but the sequence is not particularly limited. Examples of the expression control DNA actually having substitution or insertion modification or mutation upstream of the R-form specific enoyl CoA hydratase gene include those containing the nucleotide sequence shown in any of SEQ ID NOs: 37 to 45. It is not limited to these.
- the present invention further includes any one of a promoter consisting of the base sequence represented by SEQ ID NOs: 47 to 50 or a modified REP comprising the base sequence represented by any of SEQ ID NOs: 55 to 59 And a modified phaC1SD sequence represented by any one of SEQ ID NOs: 52 and 60 to 66 are also provided.
- the SD sequence is placed at an appropriate position downstream of the promoter.
- the above promoter, modified REP, phaC1SD sequence, modified phaC1SD sequence, DNA such as expression-regulated DNA can be produced by using, for example, an automatic nucleic acid synthesizer, and amplified by a nucleic acid amplification method such as a PCR method. can do.
- the microorganism of the present invention in which the expression of the R-form-specific enoyl CoA hydratase gene is regulated is used for production of a PHA copolymer
- the microorganism contains a PHA synthase gene in addition to the R-form-specific enoyl CoA hydratase gene. It must be contained.
- a microorganism originally having both the R-form-specific enoyl CoA hydratase gene and the PHA synthase gene may be used as the microorganism used in the present invention, or the R-form-specific enoyl CoA hydratase gene and the PHA synthase gene.
- the PHA synthase gene may be present on a chromosome or plasmid or megaplasmid originally possessed by the microorganism, or may be present on a foreign vector such as a plasmid, phage, or phagemid. However, it is preferably present on the chromosome, on the plasmid, or on the megaplasmid, and more preferably on the chromosome.
- PHA synthase gene Although it does not specifically limit as a PHA synthase gene, The gene which codes PHA synthetase which can synthesize
- a PHA synthase gene (phaC gene) possessed by a wild strain of Caviae, a gene encoding the amino acid sequence shown in SEQ ID NO: 46 which is a mutant enzyme gene thereof, and 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46 More preferred is a gene encoding a PHA synthase capable of synthesizing a copolymer PHA containing a 3HH monomer having an amino acid sequence having The sequence identity is more preferably 90% or more, still more preferably 95% or more, and still more preferably 98% or more.
- C.I. Necator As such a PHA-producing microorganism capable of synthesizing a PHA copolymer having an R-form-specific enoyl CoA hydratase gene and containing a 3HH monomer, C.I. Necator, more preferably C.I. Necator H16 shares A.
- a preferred example is a microorganism into which a PHA synthase gene comprising a base sequence encoding the amino acid sequence shown in SEQ ID NO: 46 derived from Caviae has been introduced.
- the method for producing a microorganism capable of producing a PHA copolymer containing 3HH monomer as a structural unit is not particularly limited. Examples are given below using necator as a host. A. as exemplified above. CHAAE derived PHA synthase gene C. by homologous recombination method on the genomic DNA of necator. Replace with necator PHA synthase gene. Then, an expression control DNA containing the above promoter and SD sequence is inserted into a base sequence upstream of the R-form-specific enoyl CoA hydratase gene present on the genomic DNA by a method such as homologous recombination.
- the promoter, the SD sequence, and the nucleic acid sequence selected from the expression control sequence, homology upstream and / or downstream of the introduction site of the target locus on the genomic DNA in addition to the above-mentioned gene to be introduced, the promoter, the SD sequence, and the nucleic acid sequence selected from the expression control sequence, homology upstream and / or downstream of the introduction site of the target locus on the genomic DNA.
- a target vector on the genomic DNA by transforming the above target microorganism using a known method such as the calcium chloride method, electroporation method, polyethylene glycol method, spheroplast method, etc.
- the nucleic acid element of interest can be introduced into the locus.
- the vector for example, plasmid, phage, phagemid and the like can be used.
- the vector can further include a marker gene for selecting a target microorganism, for example, a terminator such as a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene.
- a terminator such as a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene.
- the gene expression activity of the modified phaC1 promoter (modified REP) and the modified phaC1SD sequence can be evaluated by linking a reporter gene downstream of the promoter and the SD sequence and measuring the expression level of the reporter gene by a known method. it can.
- the reporter gene to be used is not particularly limited.
- genes that can measure enzyme activity such as ⁇ -galactosidase gene and acid phosphatase gene, those that can confirm gene expression level relatively and indirectly, such as fluorescent protein (GFP , EGFP, etc.) and photoprotein (luciferase, etc.).
- GFP fluorescent protein
- EGFP EGFP
- photoprotein luciferase, etc.
- a polyhydroxyalkanoate (PHA) copolymer having a controlled monomer composition ratio can be produced.
- PHA polyhydroxyalkanoate
- the expression control sequence is changed to various monomer composition ratios. can do.
- An example of a PHA copolymer is a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH), a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyoctanoic acid (3HO).
- the number of carbon atoms of the monomer is, for example, 4 to 8 or more, but is not limited thereto.
- the 3HH monomer composition ratio (mol%) in the copolymer can be controlled between about 5 and about 15.
- the 3HH monomer composition ratio (mol%) of P (3HB-co-3HH) is preferably about 6 to about 15, more preferably about 8 to about 13, and even more preferably about 9 to about
- the biodegradable polymer having high versatility and flexibility can be produced.
- the PHA copolymer is preferably a copolymer containing 3-hydroxyhexanoic acid (3HH) monomer as a structural unit, and in particular, P (3HB-co-3HH) having the above-mentioned monomer composition ratio. .
- a method for producing a polyhydroxyalkanoate copolymer The present invention also includes culturing the microorganism and recovering the polyhydroxyalkanoate (PHA) copolymer from the microorganism. A method for producing a copolymer is provided.
- the PHA copolymer is accumulated in the microbial cells by culturing a microorganism containing a PHA synthase gene whose expression is regulated as described above, and the R-form specific enoyl CoA hydratase gene is regulated. , Producing PHA copolymers.
- Any carbon source can be used as a carbon source during the culture as long as the PHA-producing microorganism of the present invention can be assimilated.
- sugars such as glucose, fructose, and sucrose, palm oil, and palm kernel oil are used.
- Palm oil and palm kernel oil which is a low melting point fraction obtained by separating palm kernel oil
- PFAD palm oil fatty acid from the viewpoint of avoiding competition with food in particular.
- PKFAD palm kernel oil fatty acid distillate
- rapeseed oil fatty acid distillate and the like.
- Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
- examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like.
- examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.
- Conditions such as culture temperature, culture time, culture pH, culture medium, etc. are used for microorganisms to be used, for example, the genus Ralstonia, the genus Capriavidus, the genus Wautersia, the genus Aeromonas, the genus Escherichia.
- the culture conditions may be those normally used in bacteria such as the genus Alcaligenes and Pseudomonas.
- the recovery of the PHA copolymer from the bacterial cells is not particularly limited, but can be performed, for example, by the following method.
- the cells are separated from the culture solution with a centrifuge, and the cells are washed with distilled water and methanol and dried.
- a PHA copolymer is extracted from the dried cells using an organic solvent such as chloroform.
- the bacterial cell component is removed from the organic solvent solution containing the PHA copolymer by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the PHA copolymer. Further, the supernatant is removed by filtration or centrifugation and dried to recover the PHA copolymer.
- the analysis of the weight average molecular weight (Mw) of the obtained PHA copolymer and the monomer composition (mol%) such as 3HH can be performed by, for example, a gas chromatography method, a nuclear magnetic resonance method, or the like.
- the PHA copolymer produced in the present invention is preferably a PHA copolymer containing 3HH monomer as a structural unit, more preferably 3HB monomer and P (3HB-co-3HH) having 3HH monomer as a structural unit.
- P (3HB-co-3HH) having a composition ratio of 5 mol% or more and 15 mol% or less is more preferable.
- the PHA obtained in the present invention has a controlled monomer ratio such as 3HH composition, and can have a wide range of physical properties applicable from hard polymers to soft polymers.
- the overall genetic manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
- enzymes, cloning hosts, etc. used for gene manipulation can be purchased from market suppliers and used according to the explanation.
- the enzyme is not particularly limited as long as it can be used for gene manipulation.
- PCR was performed using the primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 using the chromosomal DNA of necator H16 strain as a template. PCR was (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 2 minutes for 25 cycles, and the polymerase used was KOD-plus- (Toyobo). . Similarly, PCR was performed using the primers represented by SEQ ID NO: 3 and SEQ ID NO: 4.
- PCR was performed under the same conditions using the two DNA fragments obtained by the above PCR as templates and using the primers represented by SEQ ID NOs: 1 and 4, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with SwaI-digested vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), and nucleotide sequences upstream and downstream of the phaZ6 structural gene.
- a plasmid vector pNS2X-phaZ6 (-+) for gene disruption having
- PCR was performed under the same conditions using the primers shown by SEQ ID NO: 5 and SEQ ID NO: 6 using the chromosomal DNA of necator H16 strain as a template. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 7 and SEQ ID NO: 8. Furthermore, PCR was performed under the same conditions using the two DNA fragments obtained by the above PCR as templates and using the primers shown in SEQ ID NOs: 5 and 8, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708 digested with SwaI with DNA ligase (Ligation High (manufactured by Toyobo)), and the DNA sequence upstream and downstream from the phaZ1 structural gene.
- a plasmid vector pNS2X-phaZ1 (-+) for gene disruption having
- PCR was performed under the same conditions using the primers represented by SEQ ID NO: 9 and SEQ ID NO: 10 using the chromosomal DNA of necator H16 strain as a template. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. Furthermore, PCR was performed under the same conditions using the two DNA fragments obtained by the above PCR as templates and using the primers shown in SEQ ID NOs: 9 and 12, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI with DNA ligase (Ligation High (manufactured by Toyobo)), and the DNA sequence upstream and downstream from the phaZ2 structural gene.
- a plasmid vector pNS2X-phaZ2 (-+) for gene disruption having
- Escherichia coli S17-1 strain (ATCC47055) is transformed with the plasmid vector pNS2X-phaZ6 (-+) for gene disruption, and mixed culture with KNK005 strain (see US7384766) on Nutrient Agar medium (manufactured by Difco) to transfer the conjugation. Went.
- the strain KNK005 was introduced with a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 46 in the Sequence Listing. Necator H16 stock.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a deletion from the start codon to the stop codon of the phaZ6 gene on the chromosome was isolated by PCR analysis. This gene-disrupted strain was designated as KNK005 ⁇ phaZ6 strain. The obtained KNK005 ⁇ phaZ6 strain was obtained from C.I.
- necator is a strain in which the phaZ6 gene from the start codon to the stop codon on the chromosome of the strain H16 is deleted and a gene encoding a PHA synthase having the amino acid sequence of SEQ ID NO: 46 in the sequence listing is introduced on the chromosome. is there.
- pNS2X-phaZ1 ( ⁇ +) was used to delete the phaZ6 gene and the phaZ1 gene from the start codon to the stop codon on the chromosome, and further, the phaZ2 gene 16 Chromosomal gene disruption strains KNK005 ⁇ phaZ1,2,6 lacking from the first codon to the stop codon were prepared.
- the obtained KNK005 ⁇ phaZ1,2,6 strain is C.I.
- the starter to stop codons of the phaZ6 gene and phaZ1 gene on the chromosome of the necator H16 strain are deleted, and further, the 16th to the stop codons of the phaZ2 gene are deleted.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 15 and SEQ ID NO: 16. Furthermore, PCR was performed under the same conditions using the plasmid pKK388-1 (manufactured by CLONTECH) as a template and the primers shown in SEQ ID NO: 17 and SEQ ID NO: 18.
- PCR was carried out under the same conditions using the three types of DNA fragments obtained by the above PCR as templates and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 16, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with SwaI-digested vector pNS2X-sacB described in JP-A-2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), and the base sequence upstream of the phaJ4b structural gene, trc
- a plasmid vector pNS2X-sacB + phaJ4bU-trc-phaJ4b for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4b structural gene sequence was prepared.
- Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4bU-trc-phaJ4b for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having the trc promoter and phaC1SD sequence represented by SEQ ID NO: 38 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis. This promoter and SD sequence-inserted strain was named KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 15 and SEQ ID NO: 16. In addition, E.I. PCR was performed under the same conditions using the chromosomal DNA of E. coli strain HB101 as a template and the primers shown in SEQ ID NO: 19 and SEQ ID NO: 20.
- PCR was carried out under the same conditions using the three types of DNA fragments obtained by the above PCR as templates and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 16, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with SwaI-digested vector pNS2X-sacB described in JP-A-2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), and the base sequence upstream of the phaJ4b structural gene, lacUV5 A plasmid vector pNS2X-sacB + phaJ4bU-lacUV5-phaJ4b for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4b structural gene sequence was prepared.
- Escherichia coli S17-1 strain (ATCC47055) was transformed with the promoter and SD sequence insertion plasmid vector pNS2X-sacB + phaJ4bU-lacUV5-phaJ4b, and the KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and the Nutrient Agar medium (Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a lacUV5 promoter and a phaC1SD sequence represented by SEQ ID NO: 39 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis.
- This promoter and the SD sequence insertion strain were named KNK005 lacUV5-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 15 and SEQ ID NO: 16. Furthermore, PCR was performed under the same conditions using the plasmid pKK388-1 (manufactured by CLONTECH) as a template and the primers represented by SEQ ID NO: 17 and SEQ ID NO: 21.
- PCR was performed under the same conditions using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 16, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4b structural gene, trp A plasmid vector pNS2X-sacB + phaJ4bU-trp-phaJ4b for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4b structural gene sequence was prepared.
- Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4bU-trp-phaJ4b for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having the trp promoter and the phaC1SD sequence represented by SEQ ID NO: 40 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis.
- the promoter and the SD sequence-inserted strain were named KNK005 trp-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 15 and SEQ ID NO: 16. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 22 and SEQ ID NO: 23. PCR was performed under the same conditions using SEQ ID NO: 13 and SEQ ID NO: 16 using the three types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4b structural gene, phaC1 A plasmid vector pNS2X-sacB + phaJ4bU-REP-phaJ4b for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4b structural gene sequence was prepared.
- E. coli strain S17-1 (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4bU-REP-phaJ4b for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured in a nutrient broth medium (manufactured by Difco) for two generations, then diluted and applied onto a nutrient agar medium containing 15% sucrose, and the grown strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a phaC1 promoter and a phaC1SD sequence represented by SEQ ID NO: 37 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis.
- This promoter and the SD sequence insertion strain were named KNK005 REP-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and the primers shown in SEQ ID NO: 13 and SEQ ID NO: 14. Similarly, PCR was performed under the same conditions using the primers shown in SEQ ID NO: 24 and SEQ ID NO: 16. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 22 and SEQ ID NO: 25.
- PCR was performed under the same conditions using SEQ ID NO: 13 and SEQ ID NO: 16 using the three types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4b structural gene, phaC1 A plasmid vector pNS2X-sacB + phaJ4bU-REPSDM11-phaJ4b for DNA insertion having a promoter, a phaC1SD sequence variant, and a phaJ4b structural gene sequence was prepared.
- a promoter and SD sequence insertion strain was prepared.
- Escherichia coli S17-1 strain ATCC47055
- KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured in a nutrient broth medium (manufactured by Difco) for two generations, then diluted and applied onto a nutrient agar medium containing 15% sucrose, and the grown strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a phaC1 promoter and a phaC1SD sequence variant represented by SEQ ID NO: 41 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis.
- the promoter and SD sequence-inserted strain was named KNK005 REPSDM11-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the plasmid vector pNS2X-sacB + phaJ4bU-REPSDM11-phaJ4b prepared in Production Example 6 as a template and using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 26. Similarly, PCR was performed under the same conditions using the primers shown in SEQ ID NO: 27 and SEQ ID NO: 16. PCR was performed under the same conditions using SEQ ID NO: 13 and SEQ ID NO: 16 using the two types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4b structural gene, phaC1 Plasmid promoter pNS2X-sacB + phaJ4bU-REPN17SDM11-phaJ4b having a promoter variant, a phaC1SD sequence variant, and a phaJ4b structural gene sequence was prepared.
- Plasmid vector pNS2X-sacB + phaJ4bU-REPN17SDM11-phaJ4b for insertion of promoter and SD sequence was transformed into E. coli S17-1 strain (ATCC47055), and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a phaC1 promoter variant and a phaC1SD sequence variant represented by SEQ ID NO: 42 was isolated upstream of the phaJ4b structural gene on the chromosome by PCR analysis.
- the promoter and SD sequence-inserted strain was named KNK005 REPN17SDM11-phaJ4b / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and using the primers represented by SEQ ID NO: 28 and SEQ ID NO: 29. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 30 and SEQ ID NO: 31. Furthermore, PCR was performed under the same conditions using the plasmid pKK388-1 (manufactured by CLONTECH) as a template and the primers shown in SEQ ID NO: 32 and SEQ ID NO: 33.
- PCR was performed under the same conditions using SEQ ID NO: 28 and SEQ ID NO: 31 using the three types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708 digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4a structural gene, trc A plasmid vector pNS2X-sacB + phaJ4aU-trcSDM11-phaJ4a for DNA insertion having a promoter, a phaC1SD sequence variant, and a phaJ4a structural gene sequence was prepared.
- E. coli strain S17-1 (ATCC47055) was transformed with the promoter and SD sequence insertion plasmid vector pNS2X-sacB + phaJ4aU-trcSDM11-phaJ4a, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and the Nutrient Agar medium (Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a trc promoter and a phaC1SD sequence variant represented by SEQ ID NO: 43 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- the promoter and the SD sequence-inserted strain were named KNK005 trcSDM11-phaJ4a / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and using the primers represented by SEQ ID NO: 28 and SEQ ID NO: 29. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 34 and SEQ ID NO: 31. In addition, E.I. PCR was performed under the same conditions using the chromosomal DNA of E. coli strain HB101 as a template and the primers shown in SEQ ID NO: 35 and SEQ ID NO: 20.
- lacUV5 A plasmid vector pNS2X-sacB + phaJ4aU-lacUV5-phaJ4a for DNA insertion having a promoter, phaC1SD sequence, and phaJ4a structural gene sequence was prepared.
- E. coli strain S17-1 (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4aU-lacUV5-phaJ4a for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a lacUV5 promoter and a phaC1SD sequence represented by SEQ ID NO: 39 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- the promoter and SD sequence-inserted strain was named KNK005 lacUV5-phaJ4a / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the plasmid vector pNS2X-sacB + phaJ4aU-lacUV5-phaJ4a produced in Production Example 9 as a template and the primers shown in SEQ ID NO: 28 and SEQ ID NO: 33. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 30 and SEQ ID NO: 31. PCR was performed under the same conditions using SEQ ID NO: 28 and SEQ ID NO: 31 using the two types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with SwaI-digested vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708 and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4a structural gene, lacUV5 A plasmid vector pNS2X-sacB + phaJ4aU-lacUV5SDM11-phaJ4a for DNA insertion having a promoter, a phaC1SD sequence variant, and a phaJ4a structural gene sequence was prepared.
- Plasmid vector pNS2X-sacB + phaJ4aU-lacUV5SDM11-phaJ4a for insertion of promoter and SD sequence was transformed into Escherichia coli S17-1 strain (ATCC47055), and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a lacUV5 promoter and a phaC1SD sequence variant represented by SEQ ID NO: 44 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- the promoter and the SD sequence-inserted strain were designated as KNK005 lacUV5SDM11-phaJ4a / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and using the primers represented by SEQ ID NO: 28 and SEQ ID NO: 29. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 34 and SEQ ID NO: 31. Further, PCR was performed under the same conditions using the plasmid pKK388-1 (manufactured by CLONTECH) as a template and the primers shown in SEQ ID NO: 32 and SEQ ID NO: 21.
- PCR was carried out under the same conditions using SEQ ID NO: 28 and SEQ ID NO: 31 using the three types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4a structural gene, trp A plasmid vector pNS2X-sacB + phaJ4aU-trp-phaJ4a for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4a structural gene sequence was prepared.
- Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4aU-trp-phaJ4a for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having the trp promoter and the phaC1SD sequence represented by SEQ ID NO: 40 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- This promoter and SD sequence insertion strain was named KNK005 trp-phaJ4a / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the plasmid vector pNS2X-sacB + phaJ4aU-trp-phaJ4a produced in Production Example 11 as a template and the primers shown in SEQ ID NO: 28 and SEQ ID NO: 33. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 30 and SEQ ID NO: 31. PCR was performed under the same conditions using SEQ ID NO: 28 and SEQ ID NO: 31 using the two types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI and DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4a structural gene, trp A plasmid vector pNS2X-sacB + phaJ4aU-trpSDM11-phaJ4a for DNA insertion having a promoter, a phaC1SD sequence variant, and a phaJ4a structural gene sequence was prepared.
- Plasmid vector pNS2X-sacB + phaJ4aU-trp SDM11-phaJ4a for transformation of promoter and SD sequence was transformed into E. coli strain S17-1 (ATCC47055), and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and Nutrient Agar medium (Difco) ) The mixed culture was carried out to carry out the joint transmission.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having a trp promoter and a phaC1SD sequence variant represented by SEQ ID NO: 45 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- the promoter and the SD sequence-inserted strain were named KNK005 trpSDM11-phaJ4a / ⁇ phaZ1,2,6 strain.
- PCR was performed under the same conditions as in Production Example 1 using the chromosomal DNA of necator H16 strain as a template and using the primers represented by SEQ ID NO: 28 and SEQ ID NO: 29. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 34 and SEQ ID NO: 31. Similarly, PCR was performed under the same conditions using the primers represented by SEQ ID NO: 36 and SEQ ID NO: 23. PCR was carried out under the same conditions using SEQ ID NO: 28 and SEQ ID NO: 31 using the three types of DNA fragments obtained by the PCR as templates, and the obtained DNA fragments were digested with the restriction enzyme SwaI.
- This DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A digested with SwaI with DNA ligase (Ligation High (manufactured by Toyobo)), and the base sequence upstream of the phaJ4a structural gene, phaC1 A plasmid vector pNS2X-sacB + phaJ4aU-REP-phaJ4a for DNA insertion having a promoter, a phaC1SD sequence, and a phaJ4a structural gene sequence was prepared.
- Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + phaJ4aU-REP-phaJ4a for insertion of promoter and SD sequence, and KNK005 ⁇ phaZ1,2,6 strain prepared in Production Example 1 and the Nutrient Agar medium (manufactured by Difco) Conjugate transmission was performed by mixing culture above.
- This strain was cultured for 2 generations in Nutrient Broth medium (Difco), then diluted and applied onto Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain from which the plasmid had dropped. Furthermore, one strain having a DNA fragment having the phaC1 promoter and phaC1SD sequence represented by SEQ ID NO: 37 was isolated upstream of the phaJ4a structural gene on the chromosome by PCR analysis.
- This promoter and SD sequence insertion strain was named KNK005 REP-phaJ4a / ⁇ phaZ1,2,6 strain.
- Example 1 Production of PHA by KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 Seed medium composition is 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast- Extract, 0.9 w / v% Na 2 HPO 4 ⁇ 12H 2 O, 0.15 w / v% KH 2 PO 4 were used.
- the composition of the PHA production medium is 1.1 w / v% Na 2 HPO 4 ⁇ 12H 2 O, 0.19 w / v% KH 2 PO 4 , 0.13 w / v% (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 .7H 2 O, 0.1 v / v% trace metal salt solution (1.6 W / v% FeCl 3 .6H 2 O in 0.1 N hydrochloric acid, 1 w / v% CaCl 2 .2H 2 O, 0 0.02 w / v% CoCl 2 .6H 2 O, 0.016 w / v% CuSO 4 .5H 2 O, 0.012 w / v% NiCl 2 .6H 2 O in solution.
- palm kernel oil olein which is a low melting point fraction obtained by fractionating palm kernel oil, was used as a single carbon source.
- a glycerol stock (50 ⁇ l) of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 2 was inoculated into a seed medium (10 ml) and cultured for 24 hours, and the culture was used as a seed.
- a seed medium 10 ml
- the culture was used as a seed.
- 1.0 v / v% of the preculture seed was inoculated into a Sakaguchi flask containing 50 mL of production medium. After culturing at a culture temperature of 30 ° C. for 72 hours, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the dry cell weight was measured.
- the 3HH composition ratio of the produced polyester was measured by gas chromatography as follows. To 20 mg of the dried polyester, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added and sealed, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a polyester degradation product. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize it, and the mixture was allowed to stand until the generation of carbon dioxide gas stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography.
- the gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary column used was GL Science NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 ⁇ m). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 ⁇ l of the sample was injected. As temperature conditions, the temperature was raised from the initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at the rate of 30 ° C./min.
- the obtained polyester was copolymer polyester P (3HB-co-3HH), and the 3HH composition ratio was as shown in Table 1 above.
- Example 2 Production of PHA by KNK005 lacUV5-phaJ4b / ⁇ phaZ1,2,6 strain KNK005 lacUV5-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 3 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 3 Production of PHA by KNK005 trp-phaJ4b / ⁇ phaZ1,2,6 strain KNK005 trp-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 4 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 4 Production of PHA by KNK005 REP-phaJ4b / ⁇ phaZ1,2,6 strain KNK005 REP-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 5 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 5 Production of PHA by KNK005 REPSDM11-phaJ4b / ⁇ phaZ1,2,6 strain
- KNK005 REPSDM11-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 6 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain
- PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 6 Production of PHA by KNK005 REPN17SDM11-phaJ4b / ⁇ phaZ1,2,6 strain KNK005 REPN17SDM11-phaJ4b / ⁇ phaZ1,2,6 prepared in Production Example 7 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 7 Production of PHA using KNK005 trcSDM11-phaJ4a / ⁇ phaZ1,2,6 strain KNK005 trcSDM11-phaJ4a / ⁇ phaZ1,26 prepared in Production Example 8 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 8 Production of PHA by KNK005 lacUV5-phaJ4a / ⁇ phaZ1,2,6 strain KNK005 lacUV5-phaJ4a / ⁇ phaZ1,2,6 prepared in Production Example 9 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 9 Production of PHA by KNK005 lacUV5SDM11-phaJ4a / ⁇ phaZ1,2,6 strains Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 10 Production of PHA by KNK005 trp-phaJ4a / ⁇ phaZ1,2,6 strain KNK005 trp-phaJ4a / ⁇ phaZ1,2,6 prepared in Production Example 11 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 11 Production of PHA by KNK005 trpSDM11-phaJ4a / ⁇ phaZ1,2,6 strain KNK005 trpSDM11-phaJ4a / ⁇ phaZ1,26 prepared in Production Example 12 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured. The results are shown in Table 1 above.
- Example 12 Production of PHA by KNK005 REP-phaJ4a / ⁇ phaZ1,2,6 strain KNK005 REP-phaJ4a / ⁇ phaZ1,2,6 prepared in Production Example 13 instead of KNK005 trc-phaJ4b / ⁇ phaZ1,2,6 strain Using the strain, PHA was produced in the same manner as in Example 1, and the amount of PHA produced and the 3HH composition ratio were measured.
- Example 13 Tests and results for examining the effects of other expression control sequences on PHA production and 3HH composition ratio by microbial strains (13-1) Construction of plasmid vector pCUP2-REP-phaJ4b A plasmid vector pCUP2-REP-phaJ4b in which DNA comprising a wild type promoter (REP) and SD sequence of necator H16 strain and a base sequence containing the phaJ4b gene was inserted was prepared as follows. C. PCR was performed using the primers shown by SEQ ID NO: 72 and SEQ ID NO: 23 using the chromosomal DNA of necator H16 strain as a template. PCR was (1) 98 ° C. for 2 minutes, (2) 98 ° C.
- REP wild type promoter
- a DNA fragment obtained by PCR was designated as REP.
- C.I. PCR was performed under the same conditions using the primers represented by SEQ ID NO: 70 and SEQ ID NO: 71 using the chromosomal DNA of necator H16 strain as a template.
- the DNA fragment obtained by PCR was designated as (ReSD) -phaJ4b.
- PCR was performed under the same conditions using REP and (ReSD) -phaJ4b as templates and the primers shown in SEQ ID NO: 72 and SEQ ID NO: 71.
- the DNA fragment obtained by PCR was digested with EcoRI. This DNA fragment was designated as REP-phaJ4b (EcoRI).
- This REP-phaJ4b (EcoRI) was ligated with the plasmid vector pCUP2 described in International Publication No. WO / 2007/049716 which was cut with MunI.
- the base sequence was determined using the DNA sequencer 3130xl Genetic Analyzer made from APPLIED BIOSSYSMS, and it was the same as the base sequence of DNA used as the template. It was confirmed that the phaJ4b gene was inserted in the opposite direction to the parP gene on pCUP2.
- the obtained plasmid vector was designated as pCUP2-REP-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (13-1) above as a template and the primers shown in SEQ ID NO: 72 and SEQ ID NO: 74.
- the DNA fragment obtained by PCR was designated as REP-PU.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (13-1) above as a template and the primers shown in SEQ ID NO: 71 and SEQ ID NO: 75. It was.
- the DNA fragment obtained by PCR was designated as (REPP3M) -phaJ4b.
- PCR was carried out under the same conditions as in (13-1) above using REP-PU and (REPP3M) -phaJ4b as templates and the primers shown in SEQ ID NO: 72 and SEQ ID NO: 71.
- the DNA fragment obtained by PCR was digested with EcoRI. This DNA fragment was designated as REPP3M-phaJ4b (EcoRI).
- This REPP3M-phaJ4b (EcoRI) was ligated with the plasmid vector pCUP2 described in International Publication No. WO / 2007/049716 which was cut with MunI.
- a plasmid vector obtained by ligating REPP3M-phaJ4b (EcoRI) and pCUP2 cleaved with MunI was determined in the same manner as in (13-1) above, and the plasmid vector pCUP2 prepared in (13-1) above was determined.
- -REP-phaJ4b was obtained by introducing a mutation as shown in SEQ ID NO: 73 in the nucleotide sequence of the promoter portion of phaC1 and designated as pCUP2-REPP3M14-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (1) above as a template and the primers shown in SEQ ID NO: 71 and SEQ ID NO: 77.
- the DNA fragment obtained by PCR was designated as (REPN17) -phaJ4b.
- PCR was carried out under the same conditions as in (13-1) above using the REP-PU and (REPN17) -phaJ4b prepared in (13-2) above as a template and the primers shown in SEQ ID NO: 72 and SEQ ID NO: 71. Went.
- the DNA fragment obtained by PCR was digested with EcoRI. This DNA fragment was designated as REPN17-phaJ4b (EcoRI).
- This REPN17-phaJ4b (EcoRI) was ligated with the plasmid vector pCUP2 described in International Publication WO / 2007/049716, which was cut with MunI.
- the plasmid vector obtained by ligating REPN17-phaJ4b (EcoRI) and pCUP2 cleaved with MunI was determined in the same manner as in (13-1) above, and the plasmid vector pCUP2 prepared in (13-1) above was determined.
- -REP-phaJ4b was obtained by introducing a mutation as shown in SEQ ID NO: 76 in the nucleotide sequence of the promoter portion of phaC1 and designated as pCUP2-REPN17-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (13-1) above as a template and the primers shown in SEQ ID NO: 71 and SEQ ID NO: 78.
- the DNA fragment obtained by PCR was designated as (REP) SDM4-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (13-1) above as a template and the primers shown in SEQ ID NO: 72 and SEQ ID NO: 79. It was.
- the DNA fragment obtained by PCR was designated as REP-SDU.
- PCR was performed under the same conditions as in (13-1) above using (REP) SDM4-phaJ4b and REP-SDU as templates and using the primers represented by SEQ ID NO: 72 and SEQ ID NO: 71.
- the DNA fragment obtained by PCR was digested with EcoRI. This DNA fragment was designated as REPDMDM4-phaJ4b (EcoRI).
- This REPDM-phaJ4b (EcoRI) was ligated with the plasmid vector pCUP2 described in International Publication WO / 2007/049716, which was cut with MunI.
- the nucleotide sequence was determined in the same manner as in (13-1) above, and the plasmid vector pCUP2 prepared in (13-1) above was determined.
- -REP-phaJ4b was obtained by modifying the base sequence of the SD sequence portion of phaC1 to TGTGTGA (SEQ ID NO: 63), and designated pCUP2-REPSDM4-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the plasmid vector pCUP2-REP-phaJ4b prepared in (13-1) above as a template and the primers shown in SEQ ID NO: 71 and SEQ ID NO: 80.
- the DNA fragment obtained by PCR was designated as (REP) SDM11-phaJ4b.
- the nucleotide sequence was determined in the same manner as in (13-1) above, and the plasmid vector pCUP2 prepared in (13-1) above was determined.
- -REP-phaJ4b was obtained by modifying the base sequence of the SD sequence part of phaC1 to TCTCTCT (SEQ ID NO: 52), and designated pCUP2-REPSDM11-phaJ4b. .
- PCR was performed under the same conditions as in the above (13-1) using the chromosomal DNA of necator H16 strain as a template and the primers represented by SEQ ID NO: 81 and SEQ ID NO: 82.
- the DNA fragment obtained by PCR was digested with MunI and SpeI. This DNA fragment was designated as (ReSD) -phaJ4b (MunI, SpeI).
- This (ReSD) -phaJ4b (MunI, SpeI) was ligated with the plasmid vector pCUP2 described in International Publication No. WO / 2007/049716 digested with MunI and SpeI.
- trc The DNA fragment obtained by PCR was digested with MunI. This DNA fragment was designated as trc (MunI). Next, trc (MunI) was ligated with the plasmid pCUP2-ReSD-phaJ4b digested with MunI. For the plasmid vector in which trc (MunI) and pCUP2-ReSD-phaJ4b were digested with MunI, the nucleotide sequence was determined by the same method as in (13-1) above, and it was identical to the nucleotide sequence of the template DNA.
- trc promoter and phaJ4b gene were obtained when PCR was performed under the same conditions as in (13-1) above using the plasmid vector as a template and the primers represented by SEQ ID NO: 81 and SEQ ID NO: 83.
- a plasmid in which the trc (MunI) fragment was inserted in such a direction as to obtain an amplified fragment consisting of the contained base sequence was obtained.
- the resulting plasmid was pCUP2-trc-phaJ4b.
- phaJ4b The expression of phaJ4b is regulated with respect to the plasmid vector pCUP2-trc-phaJ4b prepared above.
- PCUP2-trcSDM1-phaJ4b in which the SD sequence of phaC1 of necator H16 strain was modified to the base sequence TGTGAGA (SEQ ID NO: 60) was prepared as follows.
- PCR was performed under the same conditions as in the above (13-1) using the chromosomal DNA of necator H16 strain as a template and the primers represented by SEQ ID NO: 81 and SEQ ID NO: 84.
- the DNA fragment obtained by PCR was digested with SpeI, and the 5 'end was phosphorylated with T4 polynucleotide kinase. This DNA fragment was designated as SDMA-phaJ4b (SpeI).
- SDMA-phaJ4b (SpeI) was ligated with the plasmid pCUP2-trc-phaJ4b of (1) in (13-6) above cut with PmaCI and SpeI.
- pCUP2-trc-phaJ4b the nucleotide sequence was determined by the same method as in (13-1) above, and (13-6) above
- the plasmid vector pCUP2-trc-phaJ4b prepared in (1) was obtained by modifying the base sequence of the SD sequence portion of phaC1 to TGTGAGA (SEQ ID NO: 60), and designated pCUP2-trcSDM1-phaJ4b.
- the base sequence of the phaC1 SD sequence portion is AGTGAGA (SEQ ID NO: 62) with respect to the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above. ) was obtained and designated as pCUP2-trcSDM3-phaJ4b.
- the base sequence of the phaC1 SD sequence portion is TGTGTGA (SEQ ID NO: 63) with respect to the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above. ) was obtained and designated as pCUP2-trcSDM4-phaJ4b.
- the base sequence of the phaC SD sequence portion is TGAGTGA (SEQ ID NO: 64) with respect to the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above. ) was obtained and designated as pCUP2-trcSDM5-phaJ4b.
- PCR was performed under the same conditions as in (13-1) above using the chromosomal DNA of necator H16 strain as a template and the primers represented by SEQ ID NO: 86 and SEQ ID NO: 81.
- the DNA fragment obtained by PCR was digested with SpeI, and the 5 ′ end was phosphorylated with T4 polynucleotide kinase. This DNA fragment was designated SDMG-phaJ4b (SpeI).
- SDMG-phaJ4b (SpeI) was ligated with the plasmid pCUP2-trc-phaJ4b of (13-6) (1) cut with PmaCI and SpeI.
- plasmid vector in which SDMG-phaJ4b (SpeI) and pCUP2-trc-phaJ4b were digested with PmaCI and SpeI the nucleotide sequence was determined in the same manner as in the above (13-1), and the above (13-6)
- the plasmid vector pCUP2-trc-phaJ4b prepared in (1) was obtained by modifying the nucleotide sequence of the SD sequence portion of phaC1 to ATATAGA (SEQ ID NO: 61), and designated pCUP2-trcSDM2-phaJ4b.
- pCUP2-trcSDM6-phaJ4b The expression of phaJ4b is regulated for the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above.
- pCUP2-trcSDM6-phaJ4b was prepared as follows by altering the SD sequence of phaC1 of necator H16 strain to the base sequence AGADATA (SEQ ID NO: 65).
- the base sequence of the phaC1 SD sequence portion was modified to AGAGATA (SEQ ID NO: 65) with respect to the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above. And obtained as pCUP2-trcSDM6-phaJ4b.
- the nucleotide sequence of the phaC SD sequence portion was changed to AGATAGA (SEQ ID NO: 66) with respect to the plasmid vector pCUP2-trc-phaJ4b prepared in (1) of (13-6) above. And obtained as pCUP2-trcSDM7-phaJ4b.
- the composition of the seed medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / V% Na 2 HPO 4 ⁇ 12H 2 O, 0.15 w / v% KH 2 PO 4 .
- kanamycin was added to a final concentration of 100 ⁇ g / ml.
- the composition of the PHA production medium is 1.1 w / v% Na 2 HPO 4 ⁇ 12H 2 O, 0.19 w / v% KH 2 PO 4 , 0.13 w / v% (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 .7H 2 O, 0.1 v / v% trace metal salt solution (1.6 W / v% FeCl 3 .6H 2 O in 0.1 N hydrochloric acid, 1 w / v% CaCl 2 .2H 2 O, 0 0.02 w / v% CoCl 2 .6H 2 O, 0.016 w / v% CuSO 4 .5H 2 O, 0.012 w / v% NiCl 2 .6H 2 O.
- palm kernel oil olein which is a low melting point fraction obtained by fractionating palm kernel oil, was used as a single carbon source.
- Plasmid vector-introduced strains obtained by transforming the above KNK005 ⁇ phaZ1,2,6 strain with each of the plasmid vectors prepared above, ie, pCUP2-REPN17-phaJ4b in KNK005 ⁇ phaZ1,2,6 strain, pCUP2-REPSDM4-phaJ4b in KNK005 ⁇ phaZ1 , 2,6, pCUP2-REPSDM11-phaJ4b in KNK005 ⁇ phaZ1,2,6, pCUP2-trcSDM1-phaJ4b in KNK005 ⁇ phaZ1,2,6, pCUP2-trcSDM3-phaJ4b in KNK005-phaCp4t6 in KNK005 ⁇ phaZ1,2,6, pCUP2-trcSDM5- haJ4b in KNK005 ⁇ phaZ1,2,6 strain, pCUP2-trcSDM2-phaJ4b in KNK005 ⁇ phaZ1,2,6 strain, pCUP2-trc
- PHA production culture was inoculated with 1.0 v / v% of the preculture seed in a Sakaguchi flask containing 50 mL of production medium. After culturing at a culture temperature of 30 ° C. for 72 hours, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the dry cell weight was measured.
- the 3HH composition ratio of the produced polyester was measured by gas chromatography as follows. To 20 mg of the dried polyester, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added and sealed, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a polyester degradation product. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize it, and the mixture was allowed to stand until the generation of carbon dioxide gas stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography.
- the gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary column used was GL Science's Neutra Bond-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 ⁇ m). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 ⁇ l of the sample was injected. As temperature conditions, the temperature was raised from the initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at the rate of 30 ° C./min.
- the obtained polyester was copolymer polyester P (3HB-co-3HH), and the 3HH composition ratio was as shown in Table 2.
- the microorganism of the present invention produces a biodegradable polymer having a wide range of physical properties from a hard polymer to a soft polymer in order to produce a polyhydroxyalkanoate (PHA) copolymer having a controlled monomer composition ratio. be able to.
- PHA polyhydroxyalkanoate
- SEQ ID NOs: 1-36, 70-72, 74-75, 77-86 Primers SEQ ID NOs: 38-40, 43-45: Expression control DNA SEQ ID NO: 60 to 66: SD SEQ ID NO: 76, 55 to 59: Promoter
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Abstract
Description
1.ゲノムDNA上にR体特異的エノイルCoAヒドラターゼ遺伝子を有し、かつ、該遺伝子の発現が調節された微生物
本発明は、ゲノムDNA上にR体特異的エノイルCoAヒドラターゼ遺伝子を有する微生物であって、該R体特異的エノイルCoAヒドラターゼ遺伝子の上流の塩基配列が、1若しくは複数の塩基の置換、欠失、挿入及び/又は付加からなる改変を含み、それにより、R体特異的エノイルCoAヒドラターゼ遺伝子の発現が調節されることを特徴とする微生物を提供する。
本発明はまた、上記微生物を培養し、該微生物からポリヒドロキシアルカノエート(PHA)共重合体を回収することを含むことを特徴とする、ポリヒドロキシアルカノエート共重合体の生産方法を提供する。
まず、遺伝子破壊用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
先ず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
プロモーター及びSD配列挿入用プラスミドベクターpNS2X-sacB+phaJ4bU-REP-phaJ4bで大腸菌S17-1株(ATCC47055)を形質転換し、製造例1で作製したKNK005ΔphaZ1,2,6株とNutrient Agar培地(Difco社製)上で混合培養して接合伝達を行った。培養液を250mg/Lのカナマイシンを含むシモンズ寒天培地(クエン酸ナトリウム2g/L、塩化ナトリウム5g/L、硫酸マグネシウム・7水塩0.2g/L、リン酸二水素アンモニウム1g/L、リン酸水素二カリウム1g/L、寒天15g/L、pH6.8)に播種し、寒天培地上で生育してきた菌株を選択して、プラスミドがKNK005ΔphaZ1,2,6株の染色体上に組み込まれた株を取得した。この株をNutrient Broth培地(Difco社製)で2世代培養した後、15%のシュークロースを含むNutrient Agar培地上に希釈して塗布し、生育してきた菌株をプラスミドが脱落した株として取得した。さらにPCRによる解析により染色体上のphaJ4b構造遺伝子の上流に、配列番号37で示される、phaC1プロモーター及びphaC1SD配列を有するDNA断片が挿入された菌株1株を単離した。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
プロモーター及びSD配列挿入用プラスミドベクターpNS2X-sacB+phaJ4bU-REPSDM11-phaJ4bで大腸菌S17-1株(ATCC47055)を形質転換し、製造例1で作製したKNK005ΔphaZ1,2,6株とNutrient Agar培地(Difco社製)上で混合培養して接合伝達を行った。培養液を250mg/Lのカナマイシンを含むシモンズ寒天培地(クエン酸ナトリウム2g/L、塩化ナトリウム5g/L、硫酸マグネシウム・7水塩0.2g/L、リン酸二水素アンモニウム1g/L、リン酸水素二カリウム1g/L、寒天15g/L、pH6.8)に播種し、寒天培地上で生育してきた菌株を選択して、プラスミドがKNK005ΔphaZ1,2,6株の染色体上に組み込まれた株を取得した。この株をNutrient Broth培地(Difco社製)で2世代培養した後、15%のシュークロースを含むNutrient Agar培地上に希釈して塗布し、生育してきた菌株をプラスミドが脱落した株として取得した。さらにPCRによる解析により染色体上のphaJ4b構造遺伝子の上流に、配列番号41で示される、phaC1プロモーター及びphaC1SD配列改変体を有するDNA断片が挿入された菌株1株を単離した。
先ず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
先ず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
まず、プロモーター及びSD配列挿入用プラスミドの作製を行った。作製は以下のように行った。
種母培地の組成は1w/v%Meat-extract、1w/v%Bacto-Trypton、0.2w/v%Yeast-extract、0.9w/v%Na2HPO4・12H2O、0.15w/v%KH2PO4とした。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例3で作製したKNK005 lacUV5-phaJ4b/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例4で作製したKNK005 trp-phaJ4b/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例5で作製したKNK005 REP-phaJ4b/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例6で作製したKNK005 REPSDM11-phaJ4b/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例7で作製したKNK005 REPN17SDM11-phaJ4b/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例8で作製したKNK005 trcSDM11-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例9で作製したKNK005 lacUV5-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例10で作製したKNK005 lacUV5SDM11-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例11で作製したKNK005 trp-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例12で作製したKNK005 trpSDM11-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例13で作製したKNK005 REP-phaJ4a/ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。
KNK005 trc-phaJ4b/ΔphaZ1,2,6株に代えて製造例1で作製したKNK005ΔphaZ1,2,6株を用いて、実施例1と同様の方法でPHAを生産し、得られたPHAの生産量、及び3HH組成比率を測定した。結果を上記の表1に示した。
(13-1)プラスミドベクターpCUP2-REP-phaJ4bの作製
C. necator H16株のphaC1の野生型のプロモーター(REP)及びSD配列と、phaJ4b遺伝子を含む塩基配列からなるDNAを挿入したプラスミドベクターpCUP2-REP-phaJ4bを次のように作製した。 C. necator H16株の染色体DNAをテンプレートとして配列番号72及び配列番号23で示されるプライマーを用いてPCRを行った。PCRは(1)98℃で2分、(2)98℃で15秒、60℃で30秒、68℃で60秒を25サイクル繰り返し、ポリメラーゼはKOD-plus-(TOYOBO製)を用いた。PCRで得たDNA断片をREPとした。次に、C. necator H16株の染色体DNAをテンプレートとして配列番号70及び配列番号71で示されるプライマーを用いて同様の条件でPCRを行った。PCRで得られたDNA断片を(ReSD)-phaJ4bとした。次に、REP及び(ReSD)-phaJ4bをテンプレートとして配列番号72及び配列番号71で示されるプライマーを用いて同様の条件でPCRを行った。PCRで得られたDNA断片をEcoRIで消化した。このDNA断片をREP-phaJ4b(EcoRI)とした。このREP-phaJ4b(EcoRI)を、国際公開公報WO/2007/049716号公報に記載のプラスミドベクターpCUP2をMunIで切断したものと連結した。REP-phaJ4b(EcoRI)とpCUP2をMunIで切断したものを連結したプラスミドベクターについて、APPLIED BIOSYSTEMS社製のDNAシークエンサー3130xl Genetic Analyzerを用いて塩基配列を決定し、鋳型としたDNAの塩基配列と同一であることを確認するとともに、phaJ4b遺伝子がpCUP2上のparP遺伝子とは逆向きに挿入されたものを取得した。取得したプラスミドベクターをpCUP2-REP-phaJ4bとした。
上記(13-1)で作製したプラスミドベクターpCUP2-REP-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1Cのプロモーターに配列番号73で示した変異を導入したpCUP2-REPP3M14-phaJ4bを次のように作製した。
上記(13-1)で作製したプラスミドベクターpCUP2-REP-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のプロモーターに配列番号76で示した変異を導入したpCUP2-REPN17-phaJ4bを次のように作製した。
上記(13-1)で作製したプラスミドベクターpCUP2-REP-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列TGTGTGA(配列番号63)に改変したpCUP2-REPSDM4-phaJ4bを次のように作製した。
上記(13-1)で作製したプラスミドベクターpCUP2-REP-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列TCTCTCT(配列番号52)に改変したpCUP2-REPSDM11-phaJ4bを次のように作製した。
(1)配列番号47で示されるtrcプロモーターと、C. necator H16株のphaC1のSD配列、phaJ4b遺伝子を含む塩基配列からなるDNAを挿入したプラスミドベクターpCUP2-trc-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列AGTGAGA(配列番号62)に改変したpCUP2-trcSDM3-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列TGTGTGA(配列番号63)に改変したpCUP2-trcSDM4-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列TGAGTGA(配列番号64)に改変したpCUP2-trcSDM5-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列ATATAGA(配列番号61)に改変したpCUP2-trcSDM2-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列AGAGATA(配列番号65)に改変したpCUP2-trcSDM6-phaJ4bを次のように作製した。
上記(13-6)の(1)で作製したプラスミドベクターpCUP2-trc-phaJ4bに対して、phaJ4bの発現調節を行うC. necator H16株のphaC1のSD配列を塩基配列AGATAGA(配列番号66)に改変したpCUP2-trcSDM7-phaJ4bを次のように作製した。
種母培地の組成は1w/v%Meat-extract、1w/v%Bacto-Trypton、0.2w/v%Yeast-extract、0.9w/v%Na2HPO4・12H2O、0.15w/v%KH2PO4とした。
配列番号38~40、43~45:発現調節DNA
配列番号60~66:SD配列
配列番号76、55~59:プロモーター
Claims (22)
- ゲノムDNA上にR体特異的エノイルCoAヒドラターゼ遺伝子を有する微生物であって、該R体特異的エノイルCoAヒドラターゼ遺伝子の上流の塩基配列が、1若しくは複数の塩基の置換、欠失、挿入及び/又は付加からなる改変を含み、それにより、R体特異的エノイルCoAヒドラターゼ遺伝子の発現が調節されることを特徴とする微生物。
- 前記改変が、R体特異的エノイルCoAヒドラターゼ遺伝子の発現を調節可能にする、プロモーター配列、シャインダルガルノ(SD)配列、又はそれらの両方の塩基配列に1若しくは複数の塩基の置換又は挿入を含む、請求項1記載の微生物。
- 前記R体特異的エノイルCoAヒドラターゼ遺伝子の上流の塩基配列が、置換又は挿入によって導入されたプロモーター配列を含む、請求項1又は2記載の微生物。
- 前記置換又は挿入の位置が、R体特異的エノイルCoAヒドラターゼ遺伝子の上流1万塩基以内である、請求項1~3のいずれか1項記載の微生物。
- さらにポリヒドロキシアルカノエート合成酵素遺伝子を有する、請求項1~4のいずれか1項記載の微生物。
- 前記ポリヒドロキシアルカノエート合成酵素遺伝子が、アエロモナス・キャビエ(Aeromonas caviae)由来の遺伝子である、請求項5記載の微生物。
- カプリアビダス(Cupriavidus)属である、請求項1~6のいずれか1項記載の微生物。
- カプリアビダス・ネカトール(Cupriavidus necator)である、請求項7記載の微生物。
- アエロモナス(Aeromonas)属である、請求項1~6のいずれか1項記載の微生物。
- アエロモナス・ヒドロフィラ(Aeromonas hydrophila)である、請求項9記載の微生物。
- 前記R体特異的エノイルCoAヒドラターゼ遺伝子の上流の塩基配列が、置換又は挿入によって導入された、大腸菌由来プロモーター、phaC1遺伝子プロモーター及びphaC1遺伝子改変プロモーターからなる群から選択される1つ以上のプロモーターを含む、請求項1~10のいずれか1項記載の微生物。
- 前記プロモーター配列の下流に置換又は挿入によって導入されたSD配列をさらに含む、請求項2~11のいずれか1項記載の微生物。
- 前記SD配列が、配列番号51又は52で示されるSD配列である、請求項12記載の微生物。
- 前記R体特異的エノイルCoAヒドラターゼ遺伝子が他の遺伝子とオペロンを形成している、請求項1~13のいずれか1項記載の微生物。
- モノマー組成比の制御されたポリヒドロキシアルカノエート共重合体を産生することができる、請求項5~14のいずれか1項記載の微生物。
- カプリアビダス・ネカトール(Cupriavidus necator)由来のphaC1遺伝子の改変プロモーターを含む30bp~900bpの発現調節DNAであって、該改変プロモーターが配列番号55~59の塩基配列からなる群から選択される塩基配列を含む、発現調節DNA。
- 前記phaC1遺伝子のシャインダルガルノ(SD)配列又はその改変SD配列をさらに含む、ただし該SD配列又は改変SD配列が、配列番号51、52、60~66のいずれかで示される塩基配列を含む、請求項16記載の発現調節DNA。
- 配列番号47~50で示される塩基配列からなるプロモーターのいずれか1つと、配列番号51、52、60~66のいずれかで示される塩基配列を含む、発現調節DNA。
- 請求項16~18のいずれか1項に記載の発現調節DNAを含む、請求項1~15のいずれか1項に記載の微生物。
- 請求項5~15及び19のいずれか1項に記載の微生物を培養し、該微生物からポリヒドロキシアルカノエート共重合体を回収することを含むことを特徴とする、ポリヒドロキシアルカノエート共重合体の生産方法。
- 前記ポリヒドロキシアルカノエート共重合体が、構成単位として3-ヒドロキシヘキサン酸(3HH)モノマーを含有する共重合体である、請求項20記載の方法。
- 前記ポリヒドロキシアルカノエート共重合体が、P(3HB-co-3HH)である、請求項21記載の方法。
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