WO2019194236A1 - Nucleic acid for controlling gene expression - Google Patents

Nucleic acid for controlling gene expression Download PDF

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WO2019194236A1
WO2019194236A1 PCT/JP2019/014846 JP2019014846W WO2019194236A1 WO 2019194236 A1 WO2019194236 A1 WO 2019194236A1 JP 2019014846 W JP2019014846 W JP 2019014846W WO 2019194236 A1 WO2019194236 A1 WO 2019194236A1
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nucleic acid
target gene
gene
vector
protein
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PCT/JP2019/014846
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French (fr)
Japanese (ja)
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光 末永
智彦 松沢
健彦 佐原
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国立研究開発法人産業技術総合研究所
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Priority to JP2020512292A priority Critical patent/JP7062315B2/en
Publication of WO2019194236A1 publication Critical patent/WO2019194236A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention relates to a nucleic acid for controlling gene expression.
  • the gene (s) of interest is often expressed in an appropriate host such as a microorganism, plant or animal using a recombinant DNA technique.
  • an appropriate host such as a microorganism, plant or animal using a recombinant DNA technique.
  • existing techniques for this purpose for example, a strong promoter sequence is added upstream of the target gene, or an expression vector having a large copy number is used.
  • organisms were used if the expression of the target gene could be additionally increased only by the step of additionally inserting a specific DNA sequence into an existing gene expression system constructed by various techniques.
  • Very useful in substance production a plurality of genes may be used in a substance production process utilizing a recombinant DNA technique using an organism as a host.
  • a cytochrome P450 enzyme that catalyzes the oxidation reaction of various substrates does not perform an enzyme reaction alone, and requires electron transfer through an electron transfer component such as an external electron donor (NADPH) and a reductase. To do.
  • Non-patent Documents 1 and 2 the process of electron transfer, that is, the shortage of the electron transfer component enzyme is often rate-determining rather than the catalytic reaction itself. Therefore, if only the electron transfer component enzyme can be increased in expression, efficient substance production will be possible, but for existing gene expression systems, the development of a control system for the presence or absence (on / off) of expression of the target gene has been Central. Therefore, development of a control system for finely controlling the gene expression level is expected.
  • Neisseria meningitidis Neisseria meningitidis has been shown to have different strain toxicity due to changes in NadA protein production depending on the number of repeats (TAAA) n present upstream of the nadA gene ( Non-patent document 4). Further, in Moraxella catarrhalis, which is a respiratory infection, the difference in pathogenicity has been confirmed by the change in the expression level of UspA2 by the number of repeats of (AGAT) n existing upstream (Non-patent Document 5).
  • An object of the present invention is to provide a nucleic acid capable of controlling the expression level of a target gene.
  • the present inventors have focused on the possibility of repetitive sequences found in pathogenic bacteria. That is, the present inventors have conceived of finding a novel repetitive sequence that can control the expression level of a target gene by additionally inserting it upstream of the target gene. In addition, the present inventors believe that if it becomes possible to adjust the expression level of the target gene downstream by increasing or decreasing the number of repeats of the found novel repetitive sequence, it can be a useful technique in a substance production process utilizing organisms. It was. Furthermore, the function of a repetitive sequence found in the aforementioned pathogenic bacteria that regulates the expression level of a downstream gene is generally verified only in an organism in which the repetitive sequence has been discovered.
  • the present invention includes:
  • the present invention provides: [1] A nucleic acid for regulating the expression level of a gene, the nucleic acid comprising the base sequence described in (i) or (ii) below: (I) 5 '-[T (G / A) ACATG (A / C) T] n-3' (In the above base sequence, n is an integer of 1 to 20), or (Ii) Regulating the expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted in the base sequence shown in (i) above and located downstream of the base sequence A base sequence that can be used.
  • the nucleic acid for regulating the expression level of the gene of the present invention in one embodiment, [2]
  • the nucleic acid according to [1] above, N in the base sequence represented by (i) 5 ′-[T (G / A) ACATG (A / C) T] n-3 ′ is an integer of 1 to 7.
  • this invention relates to the vector containing the nucleic acid as described in [3] said [1] or [2] in another aspect.
  • the vector of the present invention in one embodiment, [4] The vector according to [3] above, The vector further includes a target gene for adjusting the expression level, and the nucleic acid is arranged upstream so that the expression level of the gene can be controlled.
  • the present invention provides: [5] A host cell comprising the vector according to [3] or [4] above. Moreover, the host cell of the present invention, in one embodiment, [6] A host cell comprising the vector according to [5] above, The target gene is a foreign gene for a host cell. In another aspect, the present invention provides: [7] A method for expressing a target gene, The present invention relates to an expression method comprising the step of expressing the target gene to which the nucleic acid according to [1] or [2] is linked upstream.
  • the method for expressing the target gene of the present invention is, in one embodiment, [8] The expression method according to [7] above,
  • the nucleic acid is a nucleic acid comprising a base sequence having a number of repeats selected so that the expression level of the target gene is a desired amount.
  • the expression method of the target gene of the present invention in one embodiment, [9] The method for expressing a target gene according to [7] or [8] above, The step of expressing the target gene is a step of expressing the target gene contained in the vector according to the above [4] in a cell system or a cell-free system.
  • the expression method of the target gene of the present invention in one embodiment, [10] A method for expressing the target gene according to [7] or [8] above, The target gene is an endogenous gene in the cell; Before the step of expressing the target gene, the method further comprises the step of introducing the nucleic acid according to [1] or [2] upstream of the target gene present on the chromosome of the cell.
  • the present invention provides: [11] A method for producing a protein using a host cell, The step of promoting the expression of a gene encoding the protein in the host cell to produce the protein, wherein the host cell is upstream of the gene encoding the protein and the nucleic acid according to [1] or [2]
  • the present invention relates to a method for producing a protein, comprising the step of:
  • a method for producing a protein using the host cell of the present invention includes: [12] The method for producing the protein according to [11] above, In the step of producing the protein, the host cell contains the vector described in [4] above.
  • the method for producing a protein of the present invention includes: [13] The method for producing a protein according to [11] or [12] above,
  • the nucleic acid is a nucleic acid having a base sequence of a selected number of repeats so that the expression level of the gene becomes a desired amount.
  • the expression level of the target gene can be adjusted by additionally inserting the nucleic acid of the present invention upstream of the target gene.
  • FIG. 1 is a schematic diagram of the repetitive sequence insertion region of shotgun clones A to C obtained in Example 1 below containing repetitive sequences with different repetitive numbers found upstream of the metagenomic sequence in 3 to 5 repetitive numbers. Indicates.
  • FIG. 2 is a graph showing the difference in production amount of 2-hydroxymuconate semialdehyde of catechol 2,3-dioxygenase gene in shotgun clones A to C prepared using Escherichia coli DH10B strain host and pUC118 vector. .
  • FIG. 3 shows a comparison between the nucleic acid according to the present invention and the base sequence of the repetitive sequence in each biological species corresponding to the nucleic acid.
  • FIG. 1 is a schematic diagram of the repetitive sequence insertion region of shotgun clones A to C obtained in Example 1 below containing repetitive sequences with different repetitive numbers found upstream of the metagenomic sequence in 3 to 5 repetitive numbers. Indicates.
  • FIG. 2 is a graph showing the difference in production amount of 2-hydroxy
  • FIG. 4 shows a comparison between the nucleic acid according to the present invention and the base sequence of the repetitive sequence in each biological species corresponding to the nucleic acid.
  • FIG. 5 is a graph showing the difference in 2-hydroxymuconate semialdehyde production in E. coli JM109 strain hosts having different numbers of repeats upstream of the catechol 2,3-dioxygenase gene.
  • FIG. 6 is a graph showing differences in 2-hydroxymuconate semialdehyde production in E. coli BL21 (DE3) strain hosts having different numbers of repeats upstream of the catechol 2,3-dioxygenase gene.
  • FIG. 7 is a graph showing the difference in the production amount of o-Nitrophenol in E.
  • FIG. 8 is a graph showing the difference in the production amount of 2-hydroxymuconate semialdehyde in a Pseudomonas putida KT2440 strain host having a repeat sequence of 1 or 5 repeats upstream of the catechol 2,3-dioxygenase gene.
  • FIG. 9A is a schematic diagram showing the structure of a reporter gene and its promoter in each reporter plasmid used in Example 6 below.
  • FIG. 9B is a graph showing the activity of the secreted luciferase gene (CLuc) when the yeast YPH500 strain transformed with each reporter plasmid shown in FIG. 9A is cultured for 72 hours.
  • CLuc secreted luciferase gene
  • the present invention provides a nucleic acid for regulating the expression level of a gene.
  • the nucleic acid is a nucleic acid having the base sequence described in (i) or (ii) below: (I) 5 '-[T (G / A) ACATG (A / C) T] n-3' (In the above base sequence, n is an integer of 1 to 20), or (Ii) Regulating the expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted in the base sequence shown in (i) above and located downstream of the base sequence A base sequence that can be used.
  • the nucleic acid of the present invention comprises a base sequence consisting of 9 bases of 5 ′-[T (G / A) ACATG (A / C) T] -3 ′ (SEQ ID NO: 1) or a repetitive sequence thereof.
  • Specific examples of the base sequence constituting the nucleic acid of the present invention include 4 ′ of 5′-TGACATGAT-3 ′, 5′-TGACATGCT-3 ′, 5′-TAACATGAT-3 ′, 5′-TAACATGCT-3 ′.
  • the number of repeats in the repetitive sequence is not particularly limited as long as expression of the target gene can be regulated, and can be appropriately set depending on conditions such as the target gene and cells in which the target gene is expressed.
  • Examples of the repetitive sequence include those in which 2 to 20 nucleotide sequences represented by SEQ ID NO: 1 are linked, preferably 2 to 15, more preferably 2 to 10, and still more preferably 2 to 7 Listed can be listed.
  • the nucleic acid of the present invention does not exclude those in which more than 20 nucleotide sequences shown in SEQ ID NO: 1 are linked.
  • the preferable length of the repetitive sequence varies depending on the type of the target gene and the conditions of the cell or the like in which the target gene is expressed, and a preferable length of the repetitive sequence can be adopted as appropriate.
  • the expression level of the target gene can be controlled by inserting a nucleic acid comprising the base sequence upstream of the target gene.
  • regulating the expression level of a gene includes enhancing the expression level of the gene and suppressing the expression level of the gene.
  • the regulation of the gene expression level is to increase the expression level of the target gene.
  • a base sequence in which 4 or 5 base sequences represented by SEQ ID NO: 1 are linked is preferable. It is expected that a repetitive sequence with a linkage number of about 4 or 5 will most enhance the expression of the downstream gene, and the shorter the repetitive sequence and the longer repetitive sequence, the weaker the level of gene expression will be. A person skilled in the art can appropriately select the number of repeats of the base sequence according to the desired gene expression level of the target gene.
  • the expression level of the target gene is regulated by additionally inserting the nucleic acid of the present invention, for example, by introducing a vector containing an enzyme gene together with the nucleic acid of the present invention into a host cell such as E. coli
  • Proteins can be produced by culturing host cells or by inducing expression, and the expression level of the produced proteins can be evaluated by measuring with a microplate reader spectrophotometer or the like.
  • the expression level of the target gene according to the number of nucleic acid repeats can be evaluated by using a vector not containing the nucleic acid of the present invention and a vector containing a nucleic acid having 1 to 20 repeats.
  • the number of repeats of the nucleic acid of the present invention can be set so that the expression level of the target gene becomes a desired amount.
  • the nucleic acid of the present invention is represented as (i) 5 ′-[T (G / A) ACATG (A / C) T] n-3 ′ (wherein n is an integer of 1 to 20 in the above base sequence).
  • Nucleic acids consisting of possible base sequences are included.
  • several bases refer to, for example, 1, 2, 3, 4, 5, or 6 bases.
  • one or several nucleotides are used as long as 90% or more (preferably 95%, 98%, 99% or more) of the nucleotide sequence identity is maintained depending on the number of repetitions (n).
  • the vector of the present invention further includes a target gene for adjusting the expression level in addition to the nucleic acid described above, and the nucleic acid is arranged upstream so that the expression level of the target gene can be controlled.
  • the gene of interest is not particularly limited as long as it is a gene that produces a transcription product with a controlled expression level when placed downstream of the nucleic acid of the present invention.
  • the target gene may be a gene encoding a protein or non-coding RNA.
  • a gene encoding a protein is preferable.
  • the nucleic acid of the present invention can be inserted upstream of each gene encoding each component so as to control the expression level of the gene encoding each component in the multicomponent complex enzyme gene. By optimizing the expression level of each component, a single enzyme activity can be optimized.
  • the target gene may be a gene that is foreign to the host cell that expresses the target gene, or may be a gene that is endogenous to the host cell.
  • the target gene is a gene encoding a peptide (polypeptide), protein, non-coding RNA, functional nucleic acid or the like, and may be an artificially prepared gene such as a recombinant peptide or a recombinant protein.
  • the nucleic acid of the present invention is linked upstream of the target gene so that the target gene can be regulated.
  • the target gene can be inserted within a range of about 10 to 200 bps upstream of the target gene.
  • the expression of the target gene can be regulated, it may be arranged upstream of 200 bps.
  • it is in the range of about 10 to 150 bps upstream of the target gene.
  • the nucleic acid of the present invention may have a promoter either upstream or downstream thereof.
  • the method for introducing the nucleic acid of the present invention upstream of the target gene can be performed according to a known method.
  • the target gene is a foreign gene
  • the foreign gene can be introduced into a host cell using a vector as described below, for example.
  • the nucleic acid of the present invention can be introduced onto the chromosome of the cell by, for example, genome editing or homologous recombination technology.
  • Methods for genome editing and homologous recombination techniques for introducing the nucleic acid of the present invention into a region on the chromosome upstream of the target gene are not particularly limited and can be performed according to known methods (for example, Nature Reviews Genetics). 2001. 2: 769-779).
  • the ⁇ Red method Red / ET system, Funakoshi
  • the present invention provides a vector comprising the nucleic acid described above.
  • the vector is not particularly limited as long as the nucleic acid of the present invention and the target gene are operably linked and held together with a promoter so that the expression of the target gene can be regulated.
  • the vector that can be used in the present invention may be any of a cell-based vector that expresses a protein in a host and a cell-free vector that utilizes a protein translation system, and includes a plasmid, a viral vector, a phage, an integrated type (integrative type). ) Either a vector or an artificial chromosome may be used.
  • the integrative vector may be a vector of the type that is integrated into the genome of the host cell, and only a part thereof (such as a portion necessary for the expression of the polynucleotide of the present invention) is integrated into the host cell genome. This type of vector may be used.
  • the expression vector may be a DNA vector or an RNA vector. Although not limited to the following, for example, publicly known vectors such as pUC118 vector, pUC18 vector, pET28a vector, and pCB192 vector can be exemplified.
  • the cell-free vector include wheat-cell-free protein synthesis vectors such as an expression vector having a T7 or T3 promoter and a pEU-based plasmid having an SP6 promoter.
  • Polynucleotides other than the polynucleotide of the present invention such as elements that promote transcription (promoter, terminator, enhancer, secretion signal sequence, splicing signal, polyadenylation signal, ribosome binding sequence (SD sequence), etc.) May be included.
  • the element necessary for transcription may be any DNA sequence that exhibits transcriptional activity in the host cell, and can be appropriately selected according to the type of the host.
  • the element necessary for transcription may be operably linked to the polynucleotide of the present invention.
  • the promoter a known promoter may be selected according to the type of host and cell-free conditions. Although not limited to the following, pET vectors (Novagen), pQE vectors (Qiagen) and the like can be mentioned.
  • the vector containing the nucleic acid of the present invention can be prepared by a known vector preparation method.
  • the present invention provides a host cell comprising the vector described above.
  • a host cell expresses a regulated gene amount for a target gene located downstream of the nucleic acid when a vector containing the nucleic acid of the invention and the target gene located downstream thereof is introduced. It is not limited in the limit.
  • the nucleic acid of the present invention has been confirmed to exist in various organisms. Table 1 shows the species in which 5 ′-[T (G / A) ACATG (A / C) T] -3 ′ or a repetitive sequence thereof was confirmed.
  • Specific examples of host cells include, but are not limited to, E. coli, prokaryotic microorganisms other than E.
  • the initial base of the repetitive sequence may differ depending on the species from which it is derived (that is, one to several bases on the 5 ′ end side of the base sequence shown in SEQ ID NO: 1 may be deleted) ) And 1 to several mutations are present in the sequence as compared with the repetitive sequence of the base sequence shown in SEQ ID NO: 1.
  • the host cell containing the vector of the present invention is not particularly limited, and can be produced by a known method. Although not limited to the following, methods for transforming a host include stable gene transfer methods (eg, gene modification methods, genome editing techniques), transient gene transfer methods (eg, transient introduction of vectors), etc. Can be mentioned.
  • the present invention provides a method for expressing a target gene whose expression level is regulated.
  • the expression method includes a step of expressing a target gene to which the nucleic acid according to the present invention is linked upstream.
  • a cell line or a cell-free line can be used for expression of the target gene.
  • the expression conditions of the target gene in cell lines and cell-free lines are not particularly limited, and can be performed according to known methods.
  • a cell extract obtained by purifying a cell disruption solution as necessary, or an artificially prepared cell-free synthesis system can be used.
  • Cell synthesis systems generally contain elements necessary for translation, such as ribosomes necessary for protein synthesis, translation initiation factors, translation elongation factors, dissociation factors, and aminoacyl-tRNA synthetases.
  • various substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, creatine phosphate, and the like are added to the cell extract.
  • a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.
  • the cell extract include Escherichia coli extract, rabbit reticulocyte extract, and wheat germ extract.
  • the cell-free vector holding the target gene to which the nucleic acid according to the present invention is linked upstream expresses the target gene in the cell synthesis system as described above.
  • the nucleic acid is arranged upstream of the target gene so that the expression level of the target gene can be regulated, and the nucleic acid is selected so that the expression level of the target gene is a desired amount. It consists of a base sequence having the desired number of repeats.
  • the step of expressing the target gene can be a step of expressing the target gene in the host cell containing the vector according to the present invention.
  • the nucleic acid of the present invention and the target gene are arranged in the vector so that the expression level can be controlled, and the nucleic acid has a desired level of expression of the target gene. Consisting of a base sequence having a selected number of repeats.
  • the present invention also provides a method for producing a protein using a host cell.
  • the method for producing the protein includes a step of promoting the expression of a gene encoding the protein in the host cell and producing the protein, wherein the host cell is the above-described invention upstream of the gene encoding the protein.
  • the protein expressed by the host cell may be a protein encoded by a gene endogenous to the host cell or a protein encoded by a foreign gene.
  • the method for producing a protein using the host cell of the present invention is a method for producing a protein using a host cell containing the above-described vector of the present invention.
  • the nucleic acid of the present invention contained in the vector is derived from a nucleotide sequence having a selected number of repeats so that the expression level of the target gene is a desired amount.
  • a method for expressing a target gene in a host cell transformed with a vector containing the nucleic acid of the present invention and the target gene may be appropriately performed according to a known method depending on the vector, host cell, target gene and the like to be used. it can.
  • Example 1 Discovery of repetitive sequences that regulate the production of gene products
  • (1-1) Preparation of environmental DNA library
  • Environmental DNA extracted from activated sludge was blunt-ended using End-Repair Enzyme attached to CopyControl Fosmid Library Production Kit (EPICENTRE). Thereafter, DNA having an average chain length of 33 to 48 kb was fractionated from the gel by pulse field electrophoresis.
  • DNA was purified from the fragment excised using ⁇ -agarase (NEB). Subsequently, the purified DNA was used as an insert DNA fragment, and a ligation reaction was performed using a T4 DNA ligase to a pCC1FOS fosmid vector (EPICENTRE).
  • EPI300 EPI300 (EPICENTRE) was used as the host E. coli.
  • Escherichia coli transformed with ⁇ phage on LB agar medium containing 12.5 ⁇ g / mL chloramphenicol was selected by chloramphenicol resistance.
  • E. coli colonies were picked and cultured in 1 mL of 2 ⁇ YT medium containing 12.5 ⁇ g / mL chloramphenicol at 37 ° C. for 18 hours at 1200 rpm. Next, 200 ⁇ L of the E.
  • Fosmid DNA was extracted from a clone showing catechol-degrading activity using FosmidMAX DNA Purification Kit (EPICENTRE). Subsequently, the DNA was randomly fragmented using a DNA fragmentation apparatus (Gene machines), fractionated by agarose gel electrophoresis, and then agarose fragments containing about 2 kb to 3 kb of DNA were excised. After DNA was purified from the excised agarose fragment, the DNA fragment was smoothed using T4DNA Polymerase (Takara Bio Inc.).
  • the blunted DNA fragment was subjected to a vector ligation reaction with 50 ng of pUC118 / HincII / BAP that had been dephosphorylated, followed by ethanol precipitation and dissolution in 10 ⁇ L of sterile water.
  • a part of the prepared library was introduced into E. coli by electroporation (Gene pulser, BIO RAD) using 1 ⁇ L of ligation solution and 25 ⁇ L of host E. coli DH10B (Invitrogen).
  • a transformant (shotgun clone) was obtained by culturing overnight at 37 ° C.
  • nucleotide sequence was determined using the DNA prepared by TempliPhi as a template.
  • a sequencing reaction was performed using BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystem). PCR and purification were performed according to the attached instruction manual, and sequencing was performed using ABI3730XL (Applied Biosystem). Analysis of the base sequence of a fosmid clone exhibiting catechol-degrading activity using Artemis (Sanger Center). As a result, the function of unknown number consisting of 3, 4, and 5 repeats upstream of the catechol 2,3-dioxygenase gene It was found by chance that there was a repetitive sequence (Fig. 1).
  • the repetitive sequence was 5, -TGACATGATTAACATGATTAACATGATTGACATGATTGACATGCT-3 ′ (SEQ ID NO: 49), that is, a base sequence consisting of 5, -T (G / A) ACATG (A / C) T-3 ′.
  • Example 2 E. coli JM109 host, pUC18 vector, catechol 2,3-dioxygenase gene when using 2-hydroxymuconate semialdehyde production difference
  • PCR was performed using Shotgun clone A as a template and primers (SEQ ID NOs: 17 and 18). PCR was performed for 25 cycles in total, with treatment at 98 ° C. for 10 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 1 minute.
  • the PCR product was purified, this insert DNA was mixed with pUC18 vector treated with HindIII and XbaI, and cloning was performed using In-Fushion HD Cloning Kit (Takara Bio Inc.). DNA was collected, introduced into E. coli strain JM109 by the heat shock method, cultured on an LB agar medium containing 100 ⁇ g / mL ampicillin, 100 ⁇ M / mL IPTG, 40 ⁇ g / mL X-gal at 37 ° C. overnight. A conversion was obtained. As a result of spraying catechol, colonies showing a yellow color were selected and the nucleotide sequence was determined.
  • PUC-EDO-N3 taacatgattgacatgattgacatgct (SEQ ID NO: 51)
  • PUC-EDO-N4 taacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 52)
  • PUC-EDO-N5 tgacatgattaacatgattaacatgattgacatgattgacatGct-C49 tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 53)
  • PUC-EDO-N7 tgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgctgagatgattagattagattaga
  • each clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 ⁇ g / mL ampicillin and 100 ⁇ M / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified.
  • Example 3 E. coli BL21 (DE3) strain host, pET28a vector, catechol 2,3-dioxygenase gene production difference in 2-hydroxymuconate semialdehyde production
  • E. coli mutant strains PUC-EDO-N0, PUC-EDO-N1, PUC-EDO-N2, PUC-EDO-N3, PUC-EDO-N4, PUC-EDO-N5, and PUC-EDO used in Example 2 were used.
  • -N6 and PUC-EDO-N7 were cultured at 37 ° C. in LB medium containing 100 ⁇ g / mL ampicillin.
  • the repetitive sequence is inserted 12 bp upstream of the target gene.
  • T7 promoter and lac operator are originally present on the pET28a vector and are located upstream of the repetitive sequence.
  • This clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 ⁇ g / mL kanamycin, 34 ⁇ g / mL chloramphenicol, and 100 ⁇ M / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified.
  • Example 4 Difference in production amount of o-Nitrophenol when using E. coli JM109 host, pCB192 vector, beta-galactosidase gene
  • PCB-GAL-N2 tgacatgattgacatgct (SEQ ID NO: 50)
  • PCB-GAL-N3 taacatgattgacatgattgacatgct (SEQ ID NO: 51)
  • PCB-GAL-N4 taacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 52)
  • PCB-GAL-N6 tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 53)
  • the repetitive sequence is inserted 40 bp upstream of the target gene.
  • promoter and operator sequences do not exist on the pCB192 vector.
  • This clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 ⁇ g / mL ampicillin and 100 ⁇ M / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified.
  • ONPG o-Nitrophenyl- ⁇ -D-galactopyranoside
  • ONP o-Nitrophenol
  • Example 5 Pseudomonas putida KT2440 strain host, pMMB503EH vector, difference in production of 2-hydroxymuconate semialdehyde when using catechol 2,3-dioxygenase gene
  • E. coli mutant strains PUC-EDO-N1 and PUC-EDO-N5 used in Example 2 were cultured at 37 ° C. in an LB medium containing 100 ⁇ g / mL ampicillin.
  • the repetitive sequences (repeat numbers 1 and 5) and the catechol 2,3-dioxygenase gene are retained under the pacB503 tac promoter.
  • EC-PMM-EDO-N1 (tgacatgct; the actually used repetitive sequence is shown below)
  • EC-PMM-EDO-N5 tgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 49) were obtained.
  • the repetitive sequence is inserted 12 bp upstream of the target gene.
  • competent cells were prepared for the Pseudomonas putida KT2440 strain serving as a host.
  • the KT2440 strain was cultured for 12 hours in an LB liquid medium containing 100 ⁇ g / mL ampicillin. A part of the culture solution was re-cultured in LB liquid medium for 4 hours, and then the cells were collected and suspended in cold sterilized water. The cells were collected again by centrifugation and then suspended in chilled sterilized water. Further, a small amount of glycerol was added to the cells recovered by centrifugation, suspended, and then rapidly frozen at ⁇ 80 ° C. and stored until use.
  • EC-PMM-EDO-N1 and EC-PMM-EDO-N5 clones were cultured at 37 ° C. in an LB liquid medium containing 100 ⁇ g / mL streptomycin, and a plasmid was extracted from the cell culture medium. Plasmids PMM-EDO-N1 and PMM-EDO-N5 were introduced into Pseudomonas putida KT2440 strain by electroporation (BIO-RAD, MicroPulser). The obtained transformants were named PS-PMM-EDO-N1 and PS-PMM-EDO-N5. This clone was cultured overnight at 30 ° C.
  • Example 6 Yeast Saccharomyces cerevisiae YPH500 strain host, pCLY vector, difference in luciferin production when using luciferase gene
  • a reporter plasmid pCLY-MCS, ATTO Co., Ltd.
  • CLuc secreted luciferase gene
  • a CYC1 minimal promoter derived from the budding yeast CYC1 gene
  • a repetitive sequence is a CYC1 minimal promoter
  • a reporter plasmid in which various promoters added to the 5′-end of the gene were introduced upstream of the CLuc gene was prepared by the following method.
  • Plasmids were purified from the resulting transformants using the Monarch Plasmid Miniprep Kit (New England Biolabs), and each reporter plasmid pCLY-PCYC1min, pCLY-N1 was determined by determining the nucleotide sequence by Sanger sequencing (Eurofin Genomics).
  • -PCYC1min (tgacatgct; shows the repetitive sequence actually used; the same applies below)
  • pCLY-N2-PCYC1min tgacatgattgacatgct (SEQ ID NO: 50)
  • pCLY-N3-PCYC1min taacatgattgacatgattgacatgct (SEQ ID NO: 51)
  • the YPH500 strain was transformed as a host yeast strain. Transformation was performed based on the product manual using Frozen-EZ Yeast Transformation II Kit (Zymo Research).
  • the transformed cells were prepared using a plate synthesis medium (SC-ura plate medium: 6.7 g / L Yeast Nitrogen Base without Amino Acids [Difco], 1.92 g / L Yeast Synthetic Drop-out Media Supplements without uracil [Sigma-Aldrich], 2 (w / v)% D-Glucose [Sigma-Aldrich], 2 (w / v)% Bacto Agar [Difco]) and inoculated at 30 ° C. for 3 days.
  • SC-ura plate medium 6.7 g / L Yeast Nitrogen Base without Amino Acids [Difco], 1.92 g / L Yeast Synthetic Drop-out Media Supplements without uracil [Sigma-Aldrich], 2 (w / v)% D-Gluco
  • Each transformant obtained (3 colonies) was used for reporter assay experiments.
  • Each transformant produced above was cultured using a liquid synthetic medium (SC-ura + PPB liquid medium).
  • the composition of this liquid medium is 6.7 g / L Yeast Nitrogen Base without Amino Acids (Difco), 1.92 g / L Yeast Synthetic Drop-out Media Supplements without uracil (Sigma-Aldrich), 0.2 M Potassium phosphate buffer (pH 6.0) 2 (w / v)% D-Glucose (Sigma-Aldrich).
  • the medium absorbance (OD600) As for the medium absorbance (OD600), 200 ⁇ L of the main culture solution was dispensed into a 96-well transparent microplate, and the absorbance at 600 nm was measured using a microplate reader (Infinite 200 PRO [Tecan]). The CLuc activity was measured using a CLuc reporter assay kit (Ato Corporation) based on the product manual. 25 ⁇ L of the main culture solution was dispensed into a 96-well black microplate, 75 ⁇ L of a CLuc luminescent substrate solution was added, and luminescence for 5 seconds was measured using a microplate reader (Infinite 200 PRO). The obtained CLuc activity value was divided by the absorbance of the medium (OD600) to perform normalization according to the amount of cells.

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Abstract

The present invention provides a nucleic acid with which the expression amount of a target gene can be controlled. The nucleic acid according to the present invention comprises (i) a base sequence represented by 5'-[T(G/A)ACATG(A/C)T]n-3' (in the base acid sequence, n is an integer of 1-20) or (ii) a base sequence obtained by substituting, adding or deleting at least one base in the aforementioned base sequence. The expression amount of a gene positioned downstream on the base sequence can be adjusted.

Description

遺伝子発現を制御するための核酸Nucleic acids to control gene expression
　本発明は、遺伝子発現を制御するための核酸に関する。 The present invention relates to a nucleic acid for controlling gene expression.
　生物を活用した物質生産プロセスにおいては、目的産物を生産するために、組換えＤＮＡ手法を用いて、対象となる遺伝子(群)を微生物や植物や動物などの適当な宿主において発現させることが多い。目的産物の生産量を増大するためには、宿主生物一個体あたりの目的遺伝子の発現量の増加を行うことが好ましい。このための既存の手法としては、たとえば目的の遺伝子の上流に強力なプロモーター配列を付加することや、コピー数の多い発現ベクターを用いることがある。 In a substance production process utilizing living organisms, in order to produce a target product, the gene (s) of interest is often expressed in an appropriate host such as a microorganism, plant or animal using a recombinant DNA technique. . In order to increase the production amount of the target product, it is preferable to increase the expression level of the target gene per host organism. As existing techniques for this purpose, for example, a strong promoter sequence is added upstream of the target gene, or an expression vector having a large copy number is used.
　ここで、様々な手法で構築された既存の遺伝子発現系に、特定のＤＮＡ配列を追加的に挿入する工程のみで、付加的に目的遺伝子の発現の増加が可能になれば、生物を用いた物質生産において非常に有用である。
　また、生物を宿主とした組換えＤＮＡ手法を活用した物質生産プロセスにおいては、複数の遺伝子を使用する場合がある。例えば、様々な基質の酸化反応を触媒するシトクロームＰ４５０酵素は、単独では酵素反応を発揮せず、外部からの電子供与体(NADPH)とレダクターゼのような電子伝達成分を介した電子伝達を必要とする。また、このような多成分複合酵素は、触媒反応それ自体よりも電子伝達の過程、つまり電子伝達成分酵素の不足が律速となることが多い(非特許文献１および２)。
　したがって、電子伝達成分酵素のみを発現増加させることができれば、効率良い物質生産が可能となるが、既存の遺伝子発現系については、目的遺伝子の発現の有無(オン・オフ)の制御系の開発が中心である。そこで、遺伝子発現量を細かく調節するための制御系の開発が期待されている。 Here, organisms were used if the expression of the target gene could be additionally increased only by the step of additionally inserting a specific DNA sequence into an existing gene expression system constructed by various techniques. Very useful in substance production.
In addition, a plurality of genes may be used in a substance production process utilizing a recombinant DNA technique using an organism as a host. For example, a cytochrome P450 enzyme that catalyzes the oxidation reaction of various substrates does not perform an enzyme reaction alone, and requires electron transfer through an electron transfer component such as an external electron donor (NADPH) and a reductase. To do. Further, in such a multi-component complex enzyme, the process of electron transfer, that is, the shortage of the electron transfer component enzyme is often rate-determining rather than the catalytic reaction itself (Non-patent Documents 1 and 2).
Therefore, if only the electron transfer component enzyme can be increased in expression, efficient substance production will be possible, but for existing gene expression systems, the development of a control system for the presence or absence (on / off) of expression of the target gene has been Central. Therefore, development of a control system for finely controlling the gene expression level is expected.
　ここで、真核生物、特にヒトを含む進化した動植物の多くのゲノム上には、しばしば同一配列が連続した繰り返し配列(反復配列)が見られる。この反復配列の機能についてはほとんど不明であるが、ハンチントン病に代表されるヒトの疾病遺伝子を中心に一部機能が明らかになっている。近年のゲノム解析技術の発展により、原核微生物にも多くの反復配列が発見されてきたが、依然としてその多くが機能不明である。しかし最近になって、一部の病原菌を中心に、反復配列およびその繰り返し数と病原性との関係が明らかにされてきた(非特許文献３)。例えば、髄膜炎菌のNeisseria meningitidisはnadA遺伝子の上流に存在する反復配列(TAAA)nの繰り返しの数によってNadAタンパクの生産量が変化することにより、菌株の毒性が異なることが示された(非特許文献４)。また、呼吸器感染症のMoraxella catarrhalisでは、上流に存在する(AGAT)nの繰り返し数がUspA2の発現量に変化を与えることにより、病原性の違いが確認されている(非特許文献５)。 Here, on many genomes of evolved animals and plants including eukaryotes, particularly humans, repeated sequences (repetitive sequences) in which the same sequence is frequently observed are often found. Although the function of this repetitive sequence is almost unknown, some functions have been clarified mainly in human disease genes represented by Huntington's disease. With the recent development of genome analysis technology, many repetitive sequences have been discovered in prokaryotic microorganisms, but many of them still have unknown functions. Recently, however, repetitive sequences and the relationship between the number of repeats and pathogenicity have been clarified mainly in some pathogenic bacteria (Non-patent Document 3). For example, Neisseria meningitidis Neisseria meningitidis has been shown to have different strain toxicity due to changes in NadA protein production depending on the number of repeats (TAAA) n present upstream of the nadA gene ( Non-patent document 4). Further, in Moraxella catarrhalis, which is a respiratory infection, the difference in pathogenicity has been confirmed by the change in the expression level of UspA2 by the number of repeats of (AGAT) n existing upstream (Non-patent Document 5).
　本発明は、目的遺伝子の発現量の制御を行うことが可能な核酸の提供を課題とする。 An object of the present invention is to provide a nucleic acid capable of controlling the expression level of a target gene.
　本発明者らは、上記課題を解決するために、病原菌で見出されている反復配列の可能性に着目した。すなわち、目的遺伝子の上流に追加的に挿入することで目的遺伝子の発現量の制御を行うことが可能な新規な反復配列を見いだすことを着想した。また本発明者らは、見出した新規反復配列の繰り返し数の増減により下流の目的遺伝子の発現量を調整することが可能になれば、生物を活用した物質生産プロセスにおいて有用な手法となり得ると考えた。
　さらに、前述の病原菌で見いだされた、下流の遺伝子の発現量を調整する反復配列は、一般的にはその反復配列が発見された生物においてのみ機能が検証されている。ここで、特定の生物種にとどまらず、様々な生物において遺伝子産物の生産量調節機能を保有する反復配列を発見できれば、本手法を様々な生物種を用いた物質生産プロセスに応用可能であると考えた。
　上記着想に基づき鋭意検討の結果、本発明者らは環境中ＤＮＡから遺伝子の発現量を制御可能な新規の反復配列を見出した。
　すなわち、本発明は以下を含む：
　本発明は、一態様において、
〔１〕　遺伝子の発現量を調節するための核酸であって、下記(ｉ)または(ｉｉ)に記載の塩基配列からなる核酸に関する：
（ｉ）５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’
　(上記塩基配列において、ｎは１～２０の整数である。)、または、
（ｉｉ）上記(ｉ)に示される塩基配列において１または数個の塩基が置換、付加、または、欠失した塩基配列からなり、かつ、前記塩基配列の下流に位置する遺伝子の発現量を調節することのできる塩基配列。
　また、本発明の遺伝子の発現量を調節するための核酸は、一実施の形態において、
〔２〕上記〔１〕に記載の核酸であって、
上記(ｉ)５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’で示される塩基配列におけるｎが、１～７の整数であることを特徴とする。
　また、本発明は、別の態様において
〔３〕上記〔１〕または〔２〕に記載の核酸を含むベクターに関する。
　また、本発明のベクターは、一実施の形態において、
〔４〕上記〔３〕に記載のベクターであって、
　前記ベクターは発現量を調整する目的遺伝子をさらに含み、前記核酸が前記遺伝子の発現量を調節可能なように上流に配置されていることを特徴とする。
　また、本発明は、別の態様において、
〔５〕上記〔３〕または〔４〕に記載のベクターを含む宿主細胞に関する。
　また、本発明の宿主細胞は、一実施の形態において、
〔６〕上記〔５〕に記載のベクターを含む宿主細胞であって、
　前記目的遺伝子が宿主細胞に対する外来遺伝子であることを特徴とする。
　また、本発明は、別の態様において、
〔７〕目的遺伝子を発現させる方法であって、
　上記〔１〕または〔２〕に記載の核酸が上流に連結された前記目的遺伝子を発現させる工程を含む、発現方法に関する。
　ここで、本発明の目的遺伝子を発現させる方法は一実施の形態において、
〔８〕上記〔７〕に記載の発現方法であって、
　前記核酸が、前記目的遺伝子の発現量が所望の量となるように選択された反復数を有する塩基配列からなる核酸であることを特徴とする。
　また、本発明の目的遺伝子の発現方法は、一実施の形態において、
〔９〕上記〔７〕または〔８〕に記載の目的遺伝子の発現方法であって、
　前記目的遺伝子を発現させる工程が、上記〔４〕に記載のベクターに含まれる前記目的遺伝子を細胞系または無細胞系において発現させる工程であることを特徴とする。
　また、本発明の目的遺伝子の発現方法は、一実施の形態において、
〔１０〕上記〔７〕または〔８〕に記載の目的遺伝子を発現させる方法であって、
　前記目的遺伝子が細胞内の内在遺伝子であり、
　前記目的遺伝子を発現させる工程の前に、上記〔１〕または〔２〕に記載の核酸を前記細胞の染色体上に存在する前記目的遺伝子の上流に導入する工程をさらに含むことを特徴とする。
　また、本発明は、別の態様において、
〔１１〕宿主細胞を用いたタンパク質の産生方法であって、
　前記宿主細胞において前記タンパク質をコードする遺伝子の発現を促し、前記タンパク質を産生させる工程であって、前記宿主細胞は前記タンパク質をコードする遺伝子の上流に上記〔１〕または〔２〕に記載の核酸を有する工程を含む、タンパク質の産生方法に関する。
　また、本発明の宿主細胞を用いたタンパク質の産生方法は、一実施の形態において、
〔１２〕上記〔１１〕に記載のタンパク質の産生方法であって、
　前記タンパク質を産生される工程において、前記宿主細胞が上記〔４〕に記載のベクターを含むものであることを特徴とする。
　また、本発明のタンパク質の産生方法は、一実施の形態において、
〔１３〕上記〔１１〕または〔１２〕に記載のタンパク質の産生方法であって、
　前記核酸が、当該遺伝子の発現量が所望の量となるように、選択された反復数の塩基配列からなる核酸であることを特徴とする。 In order to solve the above-mentioned problems, the present inventors have focused on the possibility of repetitive sequences found in pathogenic bacteria. That is, the present inventors have conceived of finding a novel repetitive sequence that can control the expression level of a target gene by additionally inserting it upstream of the target gene. In addition, the present inventors believe that if it becomes possible to adjust the expression level of the target gene downstream by increasing or decreasing the number of repeats of the found novel repetitive sequence, it can be a useful technique in a substance production process utilizing organisms. It was.
Furthermore, the function of a repetitive sequence found in the aforementioned pathogenic bacteria that regulates the expression level of a downstream gene is generally verified only in an organism in which the repetitive sequence has been discovered. Here, if it is possible to find a repetitive sequence possessing a gene product production regulation function not only in a specific species but also in various organisms, this method can be applied to a substance production process using various species. Thought.
As a result of intensive studies based on the above idea, the present inventors have found a novel repetitive sequence capable of controlling the expression level of a gene from environmental DNA.
That is, the present invention includes:
In one aspect, the present invention provides:
[1] A nucleic acid for regulating the expression level of a gene, the nucleic acid comprising the base sequence described in (i) or (ii) below:
(I) 5 '-[T (G / A) ACATG (A / C) T] n-3'
(In the above base sequence, n is an integer of 1 to 20), or
(Ii) Regulating the expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted in the base sequence shown in (i) above and located downstream of the base sequence A base sequence that can be used.
Moreover, the nucleic acid for regulating the expression level of the gene of the present invention, in one embodiment,
[2] The nucleic acid according to [1] above,
N in the base sequence represented by (i) 5 ′-[T (G / A) ACATG (A / C) T] n-3 ′ is an integer of 1 to 7.
Moreover, this invention relates to the vector containing the nucleic acid as described in [3] said [1] or [2] in another aspect.
Moreover, the vector of the present invention, in one embodiment,
[4] The vector according to [3] above,
The vector further includes a target gene for adjusting the expression level, and the nucleic acid is arranged upstream so that the expression level of the gene can be controlled.
In another aspect, the present invention provides:
[5] A host cell comprising the vector according to [3] or [4] above.
Moreover, the host cell of the present invention, in one embodiment,
[6] A host cell comprising the vector according to [5] above,
The target gene is a foreign gene for a host cell.
In another aspect, the present invention provides:
[7] A method for expressing a target gene,
The present invention relates to an expression method comprising the step of expressing the target gene to which the nucleic acid according to [1] or [2] is linked upstream.
Here, the method for expressing the target gene of the present invention is, in one embodiment,
[8] The expression method according to [7] above,
The nucleic acid is a nucleic acid comprising a base sequence having a number of repeats selected so that the expression level of the target gene is a desired amount.
Moreover, the expression method of the target gene of the present invention, in one embodiment,
[9] The method for expressing a target gene according to [7] or [8] above,
The step of expressing the target gene is a step of expressing the target gene contained in the vector according to the above [4] in a cell system or a cell-free system.
Moreover, the expression method of the target gene of the present invention, in one embodiment,
[10] A method for expressing the target gene according to [7] or [8] above,
The target gene is an endogenous gene in the cell;
Before the step of expressing the target gene, the method further comprises the step of introducing the nucleic acid according to [1] or [2] upstream of the target gene present on the chromosome of the cell.
In another aspect, the present invention provides:
[11] A method for producing a protein using a host cell,
The step of promoting the expression of a gene encoding the protein in the host cell to produce the protein, wherein the host cell is upstream of the gene encoding the protein and the nucleic acid according to [1] or [2] The present invention relates to a method for producing a protein, comprising the step of:
In addition, in one embodiment, a method for producing a protein using the host cell of the present invention includes:
[12] The method for producing the protein according to [11] above,
In the step of producing the protein, the host cell contains the vector described in [4] above.
In addition, in one embodiment, the method for producing a protein of the present invention includes:
[13] The method for producing a protein according to [11] or [12] above,
The nucleic acid is a nucleic acid having a base sequence of a selected number of repeats so that the expression level of the gene becomes a desired amount.
　本発明の核酸によれば、当該目的遺伝子の上流に追加的に本発明の核酸を挿入することで目的遺伝子の発現量の調節を行うことが可能となる。 According to the nucleic acid of the present invention, the expression level of the target gene can be adjusted by additionally inserting the nucleic acid of the present invention upstream of the target gene.
図１は、メタゲノム配列上流で発見された繰り返し数の異なる反復配列を３～５の繰り返し数で含む、下記実施例１で得られたショットガンクローンＡ～Ｃの当該反復配列挿入領域の概要図を示す。FIG. 1 is a schematic diagram of the repetitive sequence insertion region of shotgun clones A to C obtained in Example 1 below containing repetitive sequences with different repetitive numbers found upstream of the metagenomic sequence in 3 to 5 repetitive numbers. Indicates. 図２は、大腸菌DH10B株宿主およびpUC118ベクターを用いて作製されたショットガンクローンＡ～Ｃにおけるカテコール２，３－ジオキシゲナーゼ遺伝子の２－ヒドロキシムコネート　セミアルデヒドの生産量の相違を示すグラフである。FIG. 2 is a graph showing the difference in production amount of 2-hydroxymuconate semialdehyde of catechol 2,3-dioxygenase gene in shotgun clones A to C prepared using Escherichia coli DH10B strain host and pUC118 vector. . 図３は、本発明に係る核酸と、当該核酸に相当する各生物種における反復配列の塩基配列との比較を示す。FIG. 3 shows a comparison between the nucleic acid according to the present invention and the base sequence of the repetitive sequence in each biological species corresponding to the nucleic acid. 図４は、本発明に係る核酸と、当該核酸に相当する各生物種における反復配列の塩基配列との比較を示す。FIG. 4 shows a comparison between the nucleic acid according to the present invention and the base sequence of the repetitive sequence in each biological species corresponding to the nucleic acid. 図５は、カテコール２，３－ジオキシゲナーゼ遺伝子の上流に異なる繰り返し数の反復配列を有する大腸菌JM109株宿主における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違を示すグラフである。FIG. 5 is a graph showing the difference in 2-hydroxymuconate semialdehyde production in E. coli JM109 strain hosts having different numbers of repeats upstream of the catechol 2,3-dioxygenase gene. 図６は、カテコール２，３－ジオキシゲナーゼ遺伝子の上流に異なる繰り返し数の反復配列を有する大腸菌BL21(DE3)株宿主における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違を示すグラフである。FIG. 6 is a graph showing differences in 2-hydroxymuconate semialdehyde production in E. coli BL21 (DE3) strain hosts having different numbers of repeats upstream of the catechol 2,3-dioxygenase gene. 図７は、ベータガラクトシダーゼ遺伝子の上流に異なる繰り返し数の反復配列を有する大腸菌JM109株宿主におけるo-Nitrophenolの生産量の相違を示すグラフである。FIG. 7 is a graph showing the difference in the production amount of o-Nitrophenol in E. coli JM109 strain hosts having different numbers of repeats upstream of the beta galactosidase gene. 図８は、カテコール２，３－ジオキシゲナーゼ遺伝子の上流に１または５の繰り返し数の反復配列を有するPseudomonas putida KT2440株宿主における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違を示すグラフである。FIG. 8 is a graph showing the difference in the production amount of 2-hydroxymuconate semialdehyde in a Pseudomonas putida KT2440 strain host having a repeat sequence of 1 or 5 repeats upstream of the catechol 2,3-dioxygenase gene. 図９Ａは、下記実施例６で用いた各レポータープラスミドにおけるレポーター遺伝子およびそのプロモーターの構成を示す概要図である。また図９Ｂは、図９Ａで示す各レポータープラスミドにより形質転換された酵母YPH500株を、７２時間培養した際の分泌型ルシフェラーゼ遺伝子（CLuc）の活性を示すグラフである。FIG. 9A is a schematic diagram showing the structure of a reporter gene and its promoter in each reporter plasmid used in Example 6 below. FIG. 9B is a graph showing the activity of the secreted luciferase gene (CLuc) when the yeast YPH500 strain transformed with each reporter plasmid shown in FIG. 9A is cultured for 72 hours.
　本発明は、一態様において、遺伝子の発現量を調節するための核酸を提供する。ここで、当該核酸は、下記(ｉ)または(ｉｉ)に記載の塩基配列からなる核酸である：
（ｉ）５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’
　(上記塩基配列において、ｎは１～２０の整数である。)、または、
（ｉｉ）上記(ｉ)に示される塩基配列において１または数個の塩基が置換、付加、または、欠失した塩基配列からなり、かつ、前記塩基配列の下流に位置する遺伝子の発現量を調節することのできる塩基配列。 In one aspect, the present invention provides a nucleic acid for regulating the expression level of a gene. Here, the nucleic acid is a nucleic acid having the base sequence described in (i) or (ii) below:
(I) 5 '-[T (G / A) ACATG (A / C) T] n-3'
(In the above base sequence, n is an integer of 1 to 20), or
(Ii) Regulating the expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted in the base sequence shown in (i) above and located downstream of the base sequence A base sequence that can be used.
　本発明の核酸は、５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］－３’(配列番号１)の９塩基からなる塩基配列またはその反復配列を含む。本発明の核酸を構成する塩基配列としては、具体的に、５’－ＴＧＡＣＡＴＧＡＴ－３’、５’－ＴＧＡＣＡＴＧＣＴ－３’、５’－ＴＡＡＣＡＴＧＡＴ－３’、５’－ＴＡＡＣＡＴＧＣＴ－３’の４つの配列を含み、また前記４つの配列を複数回繰り返した反復配列を含む。繰り返される配列は、前記４つの配列のうち同じ配列が繰り返されてもよく、異なる配列の組み合わせにより繰り返されてもよい。また、反復配列における反復数は、目的遺伝子の発現を調節できる限りにおいて特に制限されず、目的遺伝子や当該目的遺伝子を発現させる細胞などの条件により適宜設定することができる。反復配列としては例えば、配列番号１に示される塩基配列が２～２０個連結したものを挙げることができ、好ましくは２～１５個、より好ましくは２～１０個、さらに好ましくは２～７個連結したものを挙げることができる。しかしながら、本発明の核酸は配列番号１に示される塩基配列が２０個を超えて連結したものを排除しない。好ましい反復配列の長さは目的遺伝子の種類や当該目的遺伝子を発現させる細胞等の条件により異なり、適宜好ましい反復配列の長さを採用することができる。当該塩基配列からなる核酸を、目的の遺伝子の上流に挿入することで当該目的遺伝子の発現量を調節することができる。
　本明細書において、遺伝子の発現量を調節するとは、遺伝子の発現量を亢進させること、および、遺伝子の発現量を抑制することを含む。好ましい一実施の形態において、遺伝子の発現量の調節は、目的の遺伝子の発現量を亢進させることである。また、遺伝子発現量の調節の結果、通常の条件では不溶化するタンパク質を可溶化させることも可能である。
　目的遺伝子の発現量を亢進させる場合、一実施の形態において、配列番号１に示される塩基配列が４または５個連結した塩基配列(反復数３または４の塩基配列)が好ましい。４、５個程度の連結数となる反復配列が下流の遺伝子の発現を最も亢進し、それよりも短い反復配列および長い反復配列になるほど遺伝子発現の亢進の程度が弱くなることが予想される。当業者であれば、目的遺伝子の所望する遺伝子発現量に応じて、塩基配列の反復数を適宜選択することができる。 The nucleic acid of the present invention comprises a base sequence consisting of 9 bases of 5 ′-[T (G / A) ACATG (A / C) T] -3 ′ (SEQ ID NO: 1) or a repetitive sequence thereof. Specific examples of the base sequence constituting the nucleic acid of the present invention include 4 ′ of 5′-TGACATGAT-3 ′, 5′-TGACATGCT-3 ′, 5′-TAACATGAT-3 ′, 5′-TAACATGCT-3 ′. And a repetitive sequence in which the four sequences are repeated a plurality of times. Of the four sequences, the same sequence may be repeated or a combination of different sequences may be repeated. In addition, the number of repeats in the repetitive sequence is not particularly limited as long as expression of the target gene can be regulated, and can be appropriately set depending on conditions such as the target gene and cells in which the target gene is expressed. Examples of the repetitive sequence include those in which 2 to 20 nucleotide sequences represented by SEQ ID NO: 1 are linked, preferably 2 to 15, more preferably 2 to 10, and still more preferably 2 to 7 Listed can be listed. However, the nucleic acid of the present invention does not exclude those in which more than 20 nucleotide sequences shown in SEQ ID NO: 1 are linked. The preferable length of the repetitive sequence varies depending on the type of the target gene and the conditions of the cell or the like in which the target gene is expressed, and a preferable length of the repetitive sequence can be adopted as appropriate. The expression level of the target gene can be controlled by inserting a nucleic acid comprising the base sequence upstream of the target gene.
In the present specification, regulating the expression level of a gene includes enhancing the expression level of the gene and suppressing the expression level of the gene. In a preferred embodiment, the regulation of the gene expression level is to increase the expression level of the target gene. In addition, as a result of regulating the gene expression level, it is also possible to solubilize proteins that are insolubilized under normal conditions.
In the case of increasing the expression level of the target gene, in one embodiment, a base sequence (base sequence having 3 or 4 repeats) in which 4 or 5 base sequences represented by SEQ ID NO: 1 are linked is preferable. It is expected that a repetitive sequence with a linkage number of about 4 or 5 will most enhance the expression of the downstream gene, and the shorter the repetitive sequence and the longer repetitive sequence, the weaker the level of gene expression will be. A person skilled in the art can appropriately select the number of repeats of the base sequence according to the desired gene expression level of the target gene.
　本発明の核酸を追加的に挿入することにより目的遺伝子の発現量が調節されているか否かについては、例えば、本発明の核酸とともに酵素遺伝子を含むベクターを大腸菌などの宿主細胞に導入し、当該宿主細胞の培養や発現誘導によりタンパク質を産生し、産生されたタンパク質の発現量をマイクロプレートリーダー分光光度計などで測定することにより評価することができる。このとき本発明の核酸を導入していないもの、反復数が１～２０それぞれの核酸を含むベクターを用いることで、核酸の反復数に応じた目的遺伝子の発現量を評価することができる。
　上記のような評価を行うことで、目的遺伝子の発現量が所望の量となるように本発明の核酸の反復数を設定することができる。 Regarding whether or not the expression level of the target gene is regulated by additionally inserting the nucleic acid of the present invention, for example, by introducing a vector containing an enzyme gene together with the nucleic acid of the present invention into a host cell such as E. coli, Proteins can be produced by culturing host cells or by inducing expression, and the expression level of the produced proteins can be evaluated by measuring with a microplate reader spectrophotometer or the like. At this time, the expression level of the target gene according to the number of nucleic acid repeats can be evaluated by using a vector not containing the nucleic acid of the present invention and a vector containing a nucleic acid having 1 to 20 repeats.
By performing the evaluation as described above, the number of repeats of the nucleic acid of the present invention can be set so that the expression level of the target gene becomes a desired amount.
　本発明の核酸は、(ｉ)５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’ (前記塩基配列において、ｎは１～２０の整数である。)として示される塩基配列(または反復配列)において、１または数個の塩基が置換、付加、または、欠失した塩基配列からなり、かつ、前記塩基配列の下流に位置する遺伝子の発現量を調節することのできる塩基配列からなる核酸を含む。
　ここで、数個の塩基とは、例えば１個、２個、３個、４個、５個、または、６個の塩基を指す。一実施の形態においては、反復数(ｎ)に応じて９０％以上(好ましくは、９５％、９８％、９９％以上)の塩基配列の同一性を保持する範囲内で、１または数個の塩基が置換、付加、または、欠失することができる。例えば、反復数が４(ｎ＝４)のとき、１個、２個、または、３個の範囲内で塩基が置換、付加、または、欠失してもよい。 The nucleic acid of the present invention is represented as (i) 5 ′-[T (G / A) ACATG (A / C) T] n-3 ′ (wherein n is an integer of 1 to 20 in the above base sequence). The expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted, and located downstream of the base sequence. Nucleic acids consisting of possible base sequences are included.
Here, several bases refer to, for example, 1, 2, 3, 4, 5, or 6 bases. In one embodiment, one or several nucleotides are used as long as 90% or more (preferably 95%, 98%, 99% or more) of the nucleotide sequence identity is maintained depending on the number of repetitions (n). Bases can be substituted, added, or deleted. For example, when the number of repeats is 4 (n = 4), the base may be substituted, added, or deleted within the range of 1, 2, or 3.
　一実施の形態において、本発明のベクターは、上述の核酸に加えて発現量を調整する目的遺伝子をさらに含み、核酸が目的遺伝子の発現量を調節可能なように上流に配置されている。 In one embodiment, the vector of the present invention further includes a target gene for adjusting the expression level in addition to the nucleic acid described above, and the nucleic acid is arranged upstream so that the expression level of the target gene can be controlled.
　本明細書において目的遺伝子とは、本発明の核酸の下流に配置された際に当制御された発現量の転写産物を生じる遺伝子である限りにおいて特に制限されない。目的遺伝子は、タンパク質をコードする遺伝子であってもよいし、ノンコーディングＲＮＡであってもよい。好ましくはタンパク質をコードする遺伝子である。また、多成分複合酵素遺伝子における各成分をコードする遺伝子の発現量を制御するように、当該各成分をコードするそれぞれの遺伝子の上流に本発明の核酸を挿入することもできる。各成分の発現量を最適化することにより、単一の酵素活性を最適化可能となる。また、生合成遺伝子クラスターに属する各遺伝子の発現量を調整することにより、当該生合成遺伝子クラスターにより生合成される化合物の生産性の効率化を図ることができる。
　一実施の形態において目的遺伝子は、目的遺伝子を発現させる宿主細胞に対して外来の遺伝子でもよく、宿主細胞に内在する遺伝子であってもよい。目的遺伝子はペプチド（ポリペプチド）、タンパク質、ノンコーディングＲＮＡ、機能性核酸などをコードする遺伝子であり、組換えペプチド、組換えタンパク質などの人工的に調製された遺伝子であってもよい。
ここで本発明の核酸は、目的の遺伝子を調節可能なように目的遺伝子の上流に連結される。より具体的には、以下に制限されないが、例えば目的遺伝子の上流約１０～２００ｂｐｓの範囲内に挿入することができる。目的遺伝子の発現を調節できる限りにおいて２００ｂｐｓ以上の上流に配置されてもよい。好ましくは、目的遺伝子の上流約１０～１５０ｂｐｓの範囲内である。また、本発明の核酸は、その上流または下流のいずれにプロモーターが存在していてもよい。目的遺伝子が内在遺伝子または外来遺伝子のいずれの場合においても、目的遺伝子の上流へ本発明の核酸を導入する方法は公知の方法に従い行うことができる。目的遺伝子が外来遺伝子である場合、例えば下記に記載するようなベクターを用いて外来遺伝子を宿主細胞に導入することができる。あるいは、既知の遺伝子工学手法により、宿主細胞の染色体上に挿入することもできる。また、目的遺伝子が対象の細胞が有する内在遺伝子である場合、例えば、ゲノム編集や相同組換え技術により当該細胞の染色体上へ本発明の核酸を導入することができる。本発明の核酸を目的遺伝子の上流である染色体上の領域へ導入するためのゲノム編集や相同組み換え技術に関する手法は特に限定されず、公知の手法に準じて行うことができる（例えば、Nature Reviews Genetics. 2001. 2:769-779参照）。相同組換えを利用する手法としては、例えばλＲｅｄ法（Red/ET system, フナコシ社）を好適に用いることができる。 In the present specification, the gene of interest is not particularly limited as long as it is a gene that produces a transcription product with a controlled expression level when placed downstream of the nucleic acid of the present invention. The target gene may be a gene encoding a protein or non-coding RNA. A gene encoding a protein is preferable. Moreover, the nucleic acid of the present invention can be inserted upstream of each gene encoding each component so as to control the expression level of the gene encoding each component in the multicomponent complex enzyme gene. By optimizing the expression level of each component, a single enzyme activity can be optimized. Further, by adjusting the expression level of each gene belonging to the biosynthetic gene cluster, it is possible to improve the productivity of the compounds biosynthesized by the biosynthetic gene cluster.
In one embodiment, the target gene may be a gene that is foreign to the host cell that expresses the target gene, or may be a gene that is endogenous to the host cell. The target gene is a gene encoding a peptide (polypeptide), protein, non-coding RNA, functional nucleic acid or the like, and may be an artificially prepared gene such as a recombinant peptide or a recombinant protein.
Here, the nucleic acid of the present invention is linked upstream of the target gene so that the target gene can be regulated. More specifically, although not limited to the following, for example, it can be inserted within a range of about 10 to 200 bps upstream of the target gene. As long as the expression of the target gene can be regulated, it may be arranged upstream of 200 bps. Preferably, it is in the range of about 10 to 150 bps upstream of the target gene. Further, the nucleic acid of the present invention may have a promoter either upstream or downstream thereof. Regardless of whether the target gene is an endogenous gene or a foreign gene, the method for introducing the nucleic acid of the present invention upstream of the target gene can be performed according to a known method. When the target gene is a foreign gene, the foreign gene can be introduced into a host cell using a vector as described below, for example. Alternatively, it can be inserted into the chromosome of the host cell by known genetic engineering techniques. When the target gene is an endogenous gene of the target cell, the nucleic acid of the present invention can be introduced onto the chromosome of the cell by, for example, genome editing or homologous recombination technology. Methods for genome editing and homologous recombination techniques for introducing the nucleic acid of the present invention into a region on the chromosome upstream of the target gene are not particularly limited and can be performed according to known methods (for example, Nature Reviews Genetics). 2001. 2: 769-779). As a technique utilizing homologous recombination, for example, the λRed method (Red / ET system, Funakoshi) can be suitably used.
　本発明は一態様において、上述する核酸を含むベクターを提供する。
　本明細書においてベクターは、本発明の核酸と目的遺伝子とをプロモーターとともに作動可能なように連結して保持することにより、当該目的遺伝子の発現を調節可能なものであれば特に制限されない。本発明に用いることのできるベクターは、宿主においてタンパク質を発現させる細胞系ベクター、およびタンパク質翻訳系を利用する無細胞系ベクターのいずれであってもよく、プラスミド、ウイルスベクター、ファージ、組込み型(integrative)ベクターおよび人工染色体のいずれであってもよい。組込み型ベクターは、全体が宿主細胞のゲノムに組み込まれるタイプのベクターであってもよく、その一部（本発明のポリヌクレオチドが発現するために必要な部分など）のみが宿主細胞のゲノムに組み込まれるタイプのベクターであってもよい。発現ベクターは、ＤＮＡベクターまたはＲＮＡベクターであってもよい。以下に限定されないが、例えば、pUC118ベクター、pUC18ベクター、pET28aベクター、pCB192ベクターなど公知のベクターを挙げることができる。また、無細胞系ベクターとしては、Ｔ７またはＴ３プロモーターを有する発現ベクター、ＳＰ６プロモーターなどを有するｐＥＵ系プラスミド等の小麦無細胞タンパク質合成用ベクターなどを挙げることができる。
　ベクターには、転写を進める要素（プロモーター、ターミネーター、エンハンサー、分泌シグナル配列、スプライシングシグナル、ポリアデニル化シグナル、リボソーム結合配列（ＳＤ配列）など）、選択マーカーなどの、本発明のポリヌクレオチド以外のポリヌクレオチドが含まれていてもよい。転写に必要な要素は、宿主細胞において転写活性を示すＤＮＡ配列であればよく、宿主の種類に応じて適宜選択することができる。転写に必要な要素は、本発明のポリヌクレオチドに作動可能に連結されていればよい。
　プロモーターとしては宿主の種類や無細胞系の条件に応じて公知のプロモーターを選択すればよい。以下に限定されないが、pETベクター（Novagen社）、pQEベクター（Qiagen社）などを挙げることができる。
　また本発明の核酸を含むベクターは公知のベクターの作製方法により作製することができる。 In one aspect, the present invention provides a vector comprising the nucleic acid described above.
In the present specification, the vector is not particularly limited as long as the nucleic acid of the present invention and the target gene are operably linked and held together with a promoter so that the expression of the target gene can be regulated. The vector that can be used in the present invention may be any of a cell-based vector that expresses a protein in a host and a cell-free vector that utilizes a protein translation system, and includes a plasmid, a viral vector, a phage, an integrated type (integrative type). ) Either a vector or an artificial chromosome may be used. The integrative vector may be a vector of the type that is integrated into the genome of the host cell, and only a part thereof (such as a portion necessary for the expression of the polynucleotide of the present invention) is integrated into the host cell genome. This type of vector may be used. The expression vector may be a DNA vector or an RNA vector. Although not limited to the following, for example, publicly known vectors such as pUC118 vector, pUC18 vector, pET28a vector, and pCB192 vector can be exemplified. Examples of the cell-free vector include wheat-cell-free protein synthesis vectors such as an expression vector having a T7 or T3 promoter and a pEU-based plasmid having an SP6 promoter.
Polynucleotides other than the polynucleotide of the present invention, such as elements that promote transcription (promoter, terminator, enhancer, secretion signal sequence, splicing signal, polyadenylation signal, ribosome binding sequence (SD sequence), etc.) May be included. The element necessary for transcription may be any DNA sequence that exhibits transcriptional activity in the host cell, and can be appropriately selected according to the type of the host. The element necessary for transcription may be operably linked to the polynucleotide of the present invention.
As the promoter, a known promoter may be selected according to the type of host and cell-free conditions. Although not limited to the following, pET vectors (Novagen), pQE vectors (Qiagen) and the like can be mentioned.
Moreover, the vector containing the nucleic acid of the present invention can be prepared by a known vector preparation method.
　本発明は一態様において、上述するベクターを含む宿主細胞を提供する。
　本明細書において宿主細胞とは、本発明の核酸とその下流に位置する目的遺伝子とを含むベクターが導入された際に、当該核酸の下流に位置する目的遺伝子について調節された遺伝子量が発現する限りにおいて限定されない。本発明の核酸は、様々な生物に存在することを確認している。表１に、５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］－３’またはその反復配列が確認された生物種を示す。宿主細胞の具体例としては、以下に制限されないが、例えば、大腸菌、大腸菌以外の原核微生物、カビや担子菌などの真核微生物、植物や魚類やほ乳類などの高等生物などを挙げることができる。大腸菌のような汎用性の高い宿主にとどまらず、様々な生物種を宿主とした物質生産プロセスにおいて応用可能となることが期待される。なお、由来する生物種ごとに反復配列の最初の塩基が異なる場合があり(すなわち、配列番号１に示される塩基配列の５’末端側の塩基が１～数個欠失している場合がある)、また配列番号１に示される塩基配列の反復配列と比較して、配列中に１～数個の変異が存在する。本発明のベクターを含む宿主細胞は特に制限されず、公知の方法により作製することができる。以下に限定されないが、宿主を形質転換する方法としては、安定的遺伝子導入法（例、遺伝子改変法、ゲノム編集技術）、一過的遺伝子導入法（例、ベクターの一過的導入）などを挙げることができる。
Figure JPOXMLDOC01-appb-T000001
 In one aspect, the present invention provides a host cell comprising the vector described above.
In the present specification, a host cell expresses a regulated gene amount for a target gene located downstream of the nucleic acid when a vector containing the nucleic acid of the invention and the target gene located downstream thereof is introduced. It is not limited in the limit. The nucleic acid of the present invention has been confirmed to exist in various organisms. Table 1 shows the species in which 5 ′-[T (G / A) ACATG (A / C) T] -3 ′ or a repetitive sequence thereof was confirmed. Specific examples of host cells include, but are not limited to, E. coli, prokaryotic microorganisms other than E. coli, eukaryotic microorganisms such as molds and basidiomycetes, and higher organisms such as plants, fishes, and mammals. It is expected to be applicable not only to highly versatile hosts such as E. coli but also to material production processes using various biological species as hosts. The initial base of the repetitive sequence may differ depending on the species from which it is derived (that is, one to several bases on the 5 ′ end side of the base sequence shown in SEQ ID NO: 1 may be deleted) ) And 1 to several mutations are present in the sequence as compared with the repetitive sequence of the base sequence shown in SEQ ID NO: 1. The host cell containing the vector of the present invention is not particularly limited, and can be produced by a known method. Although not limited to the following, methods for transforming a host include stable gene transfer methods (eg, gene modification methods, genome editing techniques), transient gene transfer methods (eg, transient introduction of vectors), etc. Can be mentioned.
Figure JPOXMLDOC01-appb-T000001
　本発明は一態様において、発現量が調節された目的遺伝子の発現方法を提供する。当該発現方法は、上述の本発明に係る核酸が上流に連結された目的遺伝子を発現させる工程を含む。
　目的遺伝子の発現は、細胞系および無細胞系のいずれを使用することもできる。細胞系および無細胞系における目的遺伝子の発現条件は特に制限されず、公知の方法に準じて行うことができる。
　無細胞系における目的遺伝子の発現には、細胞破砕液を必要に応じて精製して得られる細胞抽出液、または、人工的に調製した無細胞合成系を用いることができる。細胞合成系には一般に、タンパク質合成に必要なリボソーム、翻訳開始因子、翻訳伸長因子、解離因子、アミノアシルｔＲＮＡ合成酵素等、翻訳に必要な要素が含まれている。タンパク質の合成を行う際には、この細胞抽出液に各種アミノ酸、ＡＴＰ、ＧＴＰなどのエネルギー源、クレアチンリン酸など、タンパク質の合成に必要なその他の物質を添加する。勿論、タンパク質合成の際に、別途用意したリボソームや各種因子、及び／又は各種酵素などを必要に応じて補充してもよい。また細胞抽出液としては大腸菌抽出液、ウサギ網状赤血球抽出液、小麦胚芽抽出液等が挙ることができる。本発明に係る核酸が上流に連結された目的遺伝子を保持する無細胞系ベクターは、上記のような細胞合成系において目的遺伝子を発現する。
　一実施の形態において、当該核酸は目的遺伝子の発現量を調節可能なように当該目的遺伝子の上流に配置されており、かつ、当該核酸は目的遺伝子の発現量が所望の量となるように選択された所望の反復数を有する塩基配列からなる。
　また、当該発現方法は一実施の形態において、目的遺伝子を発現させる工程は、上述の本発明に係るベクターを含む宿主細胞において目的遺伝子を発現させる工程とすることができる。
　一実施の形態において、当該ベクターには本発明の核酸と目的遺伝子とが発現量の制御を可能なように配置されており、かつ、当該核酸は目的遺伝子の発現量が所望の量となるように選択された反復数を有する塩基配列からなる。 In one aspect, the present invention provides a method for expressing a target gene whose expression level is regulated. The expression method includes a step of expressing a target gene to which the nucleic acid according to the present invention is linked upstream.
For expression of the target gene, either a cell line or a cell-free line can be used. The expression conditions of the target gene in cell lines and cell-free lines are not particularly limited, and can be performed according to known methods.
For expression of a target gene in a cell-free system, a cell extract obtained by purifying a cell disruption solution as necessary, or an artificially prepared cell-free synthesis system can be used. Cell synthesis systems generally contain elements necessary for translation, such as ribosomes necessary for protein synthesis, translation initiation factors, translation elongation factors, dissociation factors, and aminoacyl-tRNA synthetases. When protein is synthesized, various substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, creatine phosphate, and the like are added to the cell extract. Of course, a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis. Examples of the cell extract include Escherichia coli extract, rabbit reticulocyte extract, and wheat germ extract. The cell-free vector holding the target gene to which the nucleic acid according to the present invention is linked upstream expresses the target gene in the cell synthesis system as described above.
In one embodiment, the nucleic acid is arranged upstream of the target gene so that the expression level of the target gene can be regulated, and the nucleic acid is selected so that the expression level of the target gene is a desired amount. It consists of a base sequence having the desired number of repeats.
In one embodiment of the expression method, the step of expressing the target gene can be a step of expressing the target gene in the host cell containing the vector according to the present invention.
In one embodiment, the nucleic acid of the present invention and the target gene are arranged in the vector so that the expression level can be controlled, and the nucleic acid has a desired level of expression of the target gene. Consisting of a base sequence having a selected number of repeats.
　また本発明は一態様において、宿主細胞を用いたタンパク質の産生方法を提供する。本タンパク質の産生方法は、当該宿主細胞において当該タンパク質をコードする遺伝子の発現を促し、当該タンパク質を産生させる工程を含み、ここで当該宿主細胞は当該タンパク質をコードする遺伝子の上流に上述する本発明の核酸を有する。宿主細胞により発現するタンパク質は、宿主細胞に内在する遺伝子によりコードされるタンパク質としてもよいし、外来遺伝子によりコードされるタンパク質であってもよい。
　本発明の宿主細胞を用いたタンパク質の産生方法は一実施の形態において、上述する本発明のベクターを含む宿主細胞を用いたタンパク質の産生方法である。
　また、本発明のタンパク質の産生方法における一実施の形態において、ベクターに含まれる本発明の核酸は、目的遺伝子の発現量が所望の量となるように、選択された反復数を有する塩基配列からなる。
　本発明の核酸と目的遺伝子とを含むベクターにより形質転換された宿主細胞において目的遺伝子を発現させる方法は、用いるベクター、宿主細胞、目的遺伝子などに応じて、公知の方法に準じ適宜実施することができる。 In one embodiment, the present invention also provides a method for producing a protein using a host cell. The method for producing the protein includes a step of promoting the expression of a gene encoding the protein in the host cell and producing the protein, wherein the host cell is the above-described invention upstream of the gene encoding the protein. Of nucleic acids. The protein expressed by the host cell may be a protein encoded by a gene endogenous to the host cell or a protein encoded by a foreign gene.
In one embodiment, the method for producing a protein using the host cell of the present invention is a method for producing a protein using a host cell containing the above-described vector of the present invention.
In one embodiment of the protein production method of the present invention, the nucleic acid of the present invention contained in the vector is derived from a nucleotide sequence having a selected number of repeats so that the expression level of the target gene is a desired amount. Become.
A method for expressing a target gene in a host cell transformed with a vector containing the nucleic acid of the present invention and the target gene may be appropriately performed according to a known method depending on the vector, host cell, target gene and the like to be used. it can.
　以下に実施例により本発明をより詳細に説明するが、本発明はこれらの実施例により限定されるものではない。また、本明細書に引用する特許文献および非特許文献の開示内容は、参照により本明細書に組み込まれる。 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. The disclosures of patent documents and non-patent documents cited in this specification are incorporated herein by reference.
（実施例１：遺伝子産物の生産量を調整する反復配列の発見）
（１－１）環境ＤＮＡライブラリーの作製
　活性汚泥より抽出した環境ＤＮＡを、CopyControl Fosmid Library Production Kit(EPICENTRE社)に添付のEnd-Repair Enzymeを用いて末端の平滑化を行った。その後パルスフィールド電気泳動により平均鎖長３３～４８ｋｂ付近の大きさのＤＮＡをゲルから分画した。β－アガラーゼ(NEB社)を用いて切り出した断片からＤＮＡを精製した。
　次いで精製後のＤＮＡをインサートＤＮＡ断片として用い、ｐＣＣ１ＦＯＳフォスミドベクター(EPICENTRE社)へ、Ｔ４ＤＮＡリガーゼを用いてライゲーション反応を行った。ベクターライゲーション後、MaxPlank Lambda Packaging Extracts(EPICENTRE社)を用いて、in vitro packagingを行った。宿主大腸菌としてEPI300(EPICENTRE社)を使用した。１２.５μｇ／ｍＬのクロラムフェニコールを含むＬＢ寒天培地で、λファージにより形質転換した大腸菌をクロラムフェニコール耐性により選択した。
　大腸菌のコロニーを取り、１２.５μｇ／ｍＬのクロラムフェニコールを含む２×ＹＴ培地１ｍＬ中で、３７℃で１８時間、１２００ｒｐｍで培養した。ついで、このコロニーを採取した大腸菌の培養液のうち２００μＬを新しい８００μＬの２×ＹＴ培地に移して混合し、２５０ｒｐｍで振とうしながら３７℃で３０分間培養した後、１μＬのCopy Control Induction Solution(EPICENTRE社)を添加し、宿主大腸菌EPI300が保有するフォスミドベクターのコピー数を増加させた。さらに大腸菌を３７℃で２時間、１２００ｒｐｍで激しく振とうしながら培養した後、遠心分離により菌体を回収し、５０ｍＭのリン酸緩衝液(ｐＨ７.５)に再懸濁した。その後、１５０μＬのBugBuster Plus Benzoate Nuclease(Novagen社)を添加して、大腸菌の細胞を溶解させた。続いて、遠心分離により分離された上清を細胞抽出液として得た。
　次に、カテコール(東京化成社)を最終濃度が０.５ｍＭとなるように、５μＬずつ、１００μＬの細胞抽出液に添加し、２５℃において２５０ｒｐｍで混合しながら反応を行った。１時間および１６時間後に、カテコールの分解により、２－ヒドロキシムコネート　セミアルデヒドの形成により黄色を示すフォスミドクローンを選択した。 (Example 1: Discovery of repetitive sequences that regulate the production of gene products)
(1-1) Preparation of environmental DNA library Environmental DNA extracted from activated sludge was blunt-ended using End-Repair Enzyme attached to CopyControl Fosmid Library Production Kit (EPICENTRE). Thereafter, DNA having an average chain length of 33 to 48 kb was fractionated from the gel by pulse field electrophoresis. DNA was purified from the fragment excised using β-agarase (NEB).
Subsequently, the purified DNA was used as an insert DNA fragment, and a ligation reaction was performed using a T4 DNA ligase to a pCC1FOS fosmid vector (EPICENTRE). After vector ligation, in vitro packaging was performed using MaxPlank Lambda Packaging Extracts (EPICENTRE). EPI300 (EPICENTRE) was used as the host E. coli. Escherichia coli transformed with λ phage on LB agar medium containing 12.5 μg / mL chloramphenicol was selected by chloramphenicol resistance.
E. coli colonies were picked and cultured in 1 mL of 2 × YT medium containing 12.5 μg / mL chloramphenicol at 37 ° C. for 18 hours at 1200 rpm. Next, 200 μL of the E. coli culture solution from which the colonies were collected was transferred to a new 800 μL 2 × YT medium, mixed, cultured at 37 ° C. for 30 minutes while shaking at 250 rpm, and then 1 μL of Copy Control Induction Solution ( EPICENTRE) was added to increase the number of copies of the fosmid vector possessed by the host E. coli EPI300. Furthermore, after culturing Escherichia coli for 2 hours at 37 ° C. with vigorous shaking at 1200 rpm, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.5). Thereafter, 150 μL of BugBuster Plus Benzoate Nuclease (Novagen) was added to lyse E. coli cells. Subsequently, the supernatant separated by centrifugation was obtained as a cell extract.
Next, 5 μL of catechol (Tokyo Kasei Co., Ltd.) was added to 100 μL of the cell extract so that the final concentration was 0.5 mM, and the reaction was carried out at 25 ° C. while mixing at 250 rpm. After 1 hour and 16 hours, fosmid clones were selected that showed a yellow color due to the formation of 2-hydroxymuconate semialdehyde by degradation of catechol.
（１－２）塩基配列の決定
　カテコール分解活性を示すクローンより、FosmidMAX DNA Purification Kit(EPICENTRE社)を用いてフォスミドＤＮＡを抽出した。次いで、当該ＤＮＡを、ＤＮＡ断片化装置(Gene machines社)を用いてランダムに断片化し、アガロースゲル電気泳動により分画後、約２ｋｂ～３ｋｂのＤＮＡを含むアガロース断片を切り出した。切り出したアガロース断片よりＤＮＡを精製後、T4DNA Polymerase(タカラバイオ社)を用いてＤＮＡ断片の平滑化を行った。平滑化したＤＮＡ断片は、脱リン酸化処理を行った５０ｎｇのｐＵＣ１１８／ＨｉｎｃＩＩ／ＢＡＰとベクターライゲーション反応を行った後、エタノール沈殿を行い、１０μＬの滅菌水に溶解した。
　作製したライブラリーの一部をライゲーション溶液１μＬ、宿主大腸菌DH10B(Invitrogen社)２５μＬを用いて、エレクトロポレーション法(Gene pulser、BIO RAD社)により大腸菌に導入した。１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧ、４０μｇ／ｍＬのＸ－ｇａｌを含むＬＢ寒天培地で、３７℃で終夜培養し、形質転換体(ショットガンクローン)を得た。
　Q-Bot(GENETIX社)を用いて２８８個の大腸菌白コロニーをランダムにピッキングした。９６穴のプレートに分注した１２０μＬの１００μｇ／ｍＬのアンピシリンを含むＬＢ培地にそれぞれ植菌し、３７℃で終夜培養を行った。上記培養液にTempliPhiDNA Sequencing Template Amplification Kit(ＧＥヘルスケア社)を使用して、シーケンス用鋳型を調製した。ＴｅｍｐｌｉＰｈｉの反応は付属の取扱説明書に従った。 (1-2) Determination of nucleotide sequence Fosmid DNA was extracted from a clone showing catechol-degrading activity using FosmidMAX DNA Purification Kit (EPICENTRE). Subsequently, the DNA was randomly fragmented using a DNA fragmentation apparatus (Gene machines), fractionated by agarose gel electrophoresis, and then agarose fragments containing about 2 kb to 3 kb of DNA were excised. After DNA was purified from the excised agarose fragment, the DNA fragment was smoothed using T4DNA Polymerase (Takara Bio Inc.). The blunted DNA fragment was subjected to a vector ligation reaction with 50 ng of pUC118 / HincII / BAP that had been dephosphorylated, followed by ethanol precipitation and dissolution in 10 μL of sterile water.
A part of the prepared library was introduced into E. coli by electroporation (Gene pulser, BIO RAD) using 1 μL of ligation solution and 25 μL of host E. coli DH10B (Invitrogen). A transformant (shotgun clone) was obtained by culturing overnight at 37 ° C. in an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, and 40 μg / mL X-gal.
288 E. coli white colonies were randomly picked using Q-Bot (GENETIX). Each LB medium containing 120 μL of 100 μg / mL ampicillin dispensed in a 96-well plate was inoculated, and cultured at 37 ° C. overnight. A template for sequencing was prepared using TempliPhiDNA Sequencing Template Amplification Kit (GE Healthcare) in the culture solution. The reaction of TempliPhi followed the attached instruction manual.
　次に、ＴｅｍｐｌｉＰｈｉにより調製したＤＮＡを鋳型として塩基配列の決定を行った。BigDye Terminator V3.1 Cycle Sequencing Kit(Applied Biosystem社)によりシークエンス反応を行った。付属の取扱説明書に従いＰＣＲ、精製を行い、ABI3730XL(Applied Biosystem社)を用いて配列決定を行った。Artemis(Sanger Center)を用いて、カテコール分解活性を示すフォスミドクローンの塩基配列の解析を行った結果、カテコール２，３－ジオキシゲナーゼ遺伝子の上流に繰り返し数が３、４、５からなる機能未知の反復配列が存在することを偶然に見いだした(図１)。
　反復配列は、5,-TGACATGATTAACATGATTAACATGATTGACATGATTGACATGCT-3’(配列番号４９)であり、つまり5,-T(G/A)ACATG(A/C)T-3’からなる塩基配列であった。 Next, the nucleotide sequence was determined using the DNA prepared by TempliPhi as a template. A sequencing reaction was performed using BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystem). PCR and purification were performed according to the attached instruction manual, and sequencing was performed using ABI3730XL (Applied Biosystem). Analysis of the base sequence of a fosmid clone exhibiting catechol-degrading activity using Artemis (Sanger Center). As a result, the function of unknown number consisting of 3, 4, and 5 repeats upstream of the catechol 2,3-dioxygenase gene It was found by chance that there was a repetitive sequence (Fig. 1).
The repetitive sequence was 5, -TGACATGATTAACATGATTAACATGATTGACATGATTGACATGCT-3 ′ (SEQ ID NO: 49), that is, a base sequence consisting of 5, -T (G / A) ACATG (A / C) T-3 ′.
（１－３）反復配列の機能解析
　図１に示されたショットガンクローンＡ、Ｂ、Ｃを１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧを含むＬＢ液体培地で、３７℃で終夜培養した。菌体を回収したのち、５０ｍＭのリン酸緩衝液(ｐＨ７.５)で希釈したBugBuster Protein Extraction Reagent(Merck社)を用いて可溶性タンパクを抽出し、Bradford法により抽出されたタンパク質を定量した。
　得られた一定量の菌体抽出タンパク質に対し、終濃度において０.５ｍＭのカテコールを添加し、マイクロプレートリーダー分光光度計(TECAN社)により３７５ｎｍに最大吸収波長を持つ分解産物(２－ヒドロキシムコネート　セミアルデヒド)を定量した。その結果、反復配列の繰り返し数により、カテコール２，３－ジオキシゲナーゼによるカテコール分解産物の生成量が異なることを明らかとした(図２)。 (1-3) Functional analysis of repetitive sequences The shotgun clones A, B and C shown in FIG. 1 were cultured overnight at 37 ° C. in an LB liquid medium containing 100 μg / mL ampicillin and 100 μM / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent (Merck) diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified.
0.5 mM catechol at a final concentration is added to a certain amount of the cell-derived protein obtained, and a degradation product (2-hydroxymuco) having a maximum absorption wavelength at 375 nm by a microplate reader spectrophotometer (TECAN). Nate semialdehyde). As a result, it was clarified that the amount of catechol degradation products produced by catechol 2,3-dioxygenase varies depending on the number of repeated sequences (FIG. 2).
（１－４）当該反復配列の自然界における分布の探索
　当該反復配列について相同性検索を行った。ＮＣＢＩ(National Center for Biotechnology Information)が提供しているｗｅｂ上の相同性検索プログラムであるＢＬＡＳＴを利用し、塩基配列のデータベースはNucleotide collection(nr)を用いた。その結果、当該反復配列が、様々な繰り返し数において、各種生物種において検出された。その生物種の一例を表１に示す。また、特定の生物種における反復配列の例として、塩基配列(配列番号２～１６)を具体的に図３および図４に示す。図３および図４において、上段の塩基配列は配列番号１に示される塩基配列の繰り返しを比較対象として、下段の塩基配列がそれぞれの生物種において見出された反復配列を示す。 (1-4) Search for the natural distribution of the repetitive sequence A homology search was performed for the repetitive sequence. BLAST, which is a homology search program on the web provided by NCBI (National Center for Biotechnology Information), was used, and Nucleotide collection (nr) was used as the base sequence database. As a result, the repetitive sequence was detected in various species at various repeat numbers. An example of the species is shown in Table 1. In addition, as examples of repetitive sequences in a specific species, specific nucleotide sequences (SEQ ID NOs: 2 to 16) are shown in FIG. 3 and FIG. 3 and 4, the upper base sequence indicates a repetitive sequence in which the base sequence shown in SEQ ID NO: 1 is compared and the lower base sequence is found in each species.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（実施例２：大腸菌JM109株宿主、pUC18ベクター、カテコール2,3-ジオキシゲナーゼ遺伝子使用時における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違）
　まずは、実施例１で使用したショットガンクローンＡより、当該反復配列とカテコール２，３－ジオキシゲナーゼ遺伝子の配列のみをサブクローニングする実験を行った。ショットガンクローンＡを鋳型にし、プライマー(配列番号１７、１８)を用いてＰＣＲを行った。ＰＣＲは９８℃で１０秒、６０℃で５秒、７２℃で１分の処理を１サイクルとして合計２５サイクル行った。ＰＣＲ産物を精製し、このインサートＤＮＡと、HindIIIとXbaIで処理したｐＵＣ１８ベクターを混合し、In-Fushion HD Cloning Kit(タカラバイオ社)を使用してクローニングを行った。ＤＮＡを回収し、ヒートショック法で大腸菌JM109株に導入し、１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧ、４０μｇ／ｍＬのＸ－ｇａｌを含むＬＢ寒天培地で、３７℃で終夜培養し、形質転換体を得た。カテコールを噴霧した結果、黄色を呈するコロニーを選択し、塩基配列を決定した。得られたクローンは、繰り返し数５の反復配列およびその下流にカテコール２，３－ジオキシゲナーゼ遺伝子を保有していたので、PUC-EDO-N5と名付けた(繰り返し数とは反復配列において繰り返される基本単位となる塩基配列の数を意味する。すなわち、繰り返し数５は、反復配列として繰り返される塩基配列の基本単位が５つ存在することを意味する)。 (Example 2: E. coli JM109 host, pUC18 vector, catechol 2,3-dioxygenase gene when using 2-hydroxymuconate semialdehyde production difference)
First, from the shotgun clone A used in Example 1, an experiment for subcloning only the repetitive sequence and the catechol 2,3-dioxygenase gene sequence was performed. PCR was performed using Shotgun clone A as a template and primers (SEQ ID NOs: 17 and 18). PCR was performed for 25 cycles in total, with treatment at 98 ° C. for 10 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 1 minute. The PCR product was purified, this insert DNA was mixed with pUC18 vector treated with HindIII and XbaI, and cloning was performed using In-Fushion HD Cloning Kit (Takara Bio Inc.). DNA was collected, introduced into E. coli strain JM109 by the heat shock method, cultured on an LB agar medium containing 100 μg / mL ampicillin, 100 μM / mL IPTG, 40 μg / mL X-gal at 37 ° C. overnight. A conversion was obtained. As a result of spraying catechol, colonies showing a yellow color were selected and the nucleotide sequence was determined. Since the obtained clone had a repeat number of 5 and a catechol 2,3-dioxygenase gene downstream thereof, it was named PUC-EDO-N5. This means the number of base sequences as a unit, that is, a repeat number of 5 means that there are 5 basic units of the base sequence that are repeated as a repeat sequence).
　つぎに、PUC-EDO-N5において、当該配列の繰り返し数のみが異なるクローンの作成を行った。配列番号１９～３４に記載されたプライマーを使用し、PUC-EDO-N5を鋳型として、KOD Plus Mutagenesis Kit(TOYOBO社)の説明に従い実験を行った。得られた変異体の塩基配列を決定し、当該反復配列の０から１０の繰り返し数を保有していることを確認した。それぞれ、反復配列の繰り返し数に応じてPUC-EDO-N0、PUC-EDO-N1(tgacatgct;実際に使用した反復配列を示す。以下同様。)、PUC-EDO-N2(tgacatgattgacatgct (配列番号５０))、PUC-EDO-N3(taacatgattgacatgattgacatgct (配列番号５１))、PUC-EDO-N4(taacatgattaacatgattgacatgattgacatgct (配列番号５２))、PUC-EDO-N5(tgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号４９))、PUC-EDO-N6(tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号５３))、PUC-EDO-N7(tgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号５４))、PUC-EDO-N10(tgacatgattaacatgattaacatgattgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号５５))と名付けた。これらの株が保有するプラスミド上においては、目的遺伝子の１２ｂｐ上流に当該反復配列が挿入されている。また、pUC18ベクター上にはlac promoterおよびlac operatorがもともと存在しており、当該反復配列の上流に位置する。
　そこで、各クローンを１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧを含むＬＢ液体培地で、３７℃で終夜培養した。菌体を回収したのち、５０ｍＭのリン酸緩衝液(ｐＨ７.５)で希釈したBugBuster Protein Extraction Reagentを用いて可溶性タンパクを抽出し、Bradford法により抽出されたタンパク質を定量した。得られた一定量の菌体抽出タンパク質に対し、終濃度において０.５ｍＭのカテコールを添加し、マイクロプレートリーダー分光光度計により３７５ｎｍに最大吸収波長を持つ分解産物(２－ヒドロキシムコネート　セミアルデヒド)を定量した。その結果、反復配列の繰り返し数により、カテコール２，３－ジオキシゲナーゼによるカテコール分解産物の生成量が異なることを明らかとした(図５)。 Next, in PUC-EDO-N5, clones differing only in the number of repeats of the sequence were prepared. Experiments were performed according to the description of the KOD Plus Mutagenesis Kit (TOYOBO) using the primers shown in SEQ ID NOs: 19 to 34 and PUC-EDO-N5 as a template. The base sequence of the obtained mutant was determined, and it was confirmed that it possessed a repeat number of 0 to 10 of the repetitive sequence. PUC-EDO-N0, PUC-EDO-N1 (tgacatgct; the actually used repetitive sequence, the same applies hereinafter), PUC-EDO-N2 (tgacatgattgacatgct (SEQ ID NO: 50), respectively, depending on the number of repetitive sequences. ), PUC-EDO-N3 (taacatgattgacatgattgacatgct (SEQ ID NO: 51)), PUC-EDO-N4 (taacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 52)), PUC-EDO-N5 (tgacatgattaacatgattaacatgattgacatgattgacatGct-C49 tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 53)), PUC-EDO-N7 (tgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgctgagatgattagattagattaga On the plasmids possessed by these strains, the repetitive sequence is inserted 12 bp upstream of the target gene. In addition, the lac promoter and lac operator originally exist on the pUC18 vector and are located upstream of the repetitive sequence.
Therefore, each clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 μg / mL ampicillin and 100 μM / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified. A final amount of 0.5 mM catechol is added to the obtained microbial extract protein, and a degradation product (2-hydroxymuconate semialdehyde) having a maximum absorption wavelength at 375 nm by a microplate reader spectrophotometer Was quantified. As a result, it was clarified that the amount of catechol degradation products produced by catechol 2,3-dioxygenase varies depending on the number of repeated sequences (FIG. 5).
（実施例３：大腸菌BL21(DE3)株宿主、pET28aベクター、カテコール2,3-ジオキシゲナーゼ遺伝子使用時における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違）
　まずは、実施例２で使用した大腸菌変異株PUC-EDO-N0、PUC-EDO-N1、PUC-EDO-N2、PUC-EDO-N3、PUC-EDO-N4、PUC-EDO-N5、PUC-EDO-N6、PUC-EDO-N7を１００μｇ／ｍＬのアンピシリンを含むＬＢ培地で、37℃で培養した。菌体培養液よりプラスミドを抽出し、XbaIおよびHindIIIで消化することにより、当該反復配列とカテコール2,3-ジオキシゲナーゼ遺伝子を含む領域のみを切り出し、同じくXbaIおよびHindIIIで消化したpET28aベクターとligase反応を行った。ＤＮＡを回収し、ヒートショック法で大腸菌BL21(DE3)株に導入し、１００μｇ／ｍＬのカナマイシン、３４μｇ／ｍＬのクロラムフェニコール、１００μＭ／ｍＬのＩＰＴＧ、４０μｇ／ｍＬのＸ－ｇａｌを含むＬＢ寒天培地で、３７℃で終夜培養し、形質転換体を得た。カテコールを噴霧した結果、黄色を呈するコロニーを選択し、塩基配列を決定した。得られたクローンを、PET-EDO-N0、PET-EDO-N1（tgacatgct；実際に使用した反復配列を示す。以下同様。）、PET-EDO-N2（tgacatgattgacatgct (配列番号５０)）、PET-EDO-N3（taacatgattgacatgattgacatgct  (配列番号５１)）、PET-EDO-N4（taacatgattaacatgattgacatgattgacatgct  (配列番号５２)）、PET-EDO-N5（tgacatgattaacatgattaacatgattgacatgattgacatgct  (配列番号４９)）、PET-EDO-N6（tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct  (配列番号５３)）、PET-EDO-N7（tgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct  (配列番号５４)）と名付けた。これらの株が保有するプラスミド上においては、目的遺伝子の12bp上流に当該反復配列が挿入されている。また、pET28aベクター上にはT7 promoterおよびlac operatorがもともと存在しており、当該反復配列の上流に位置する。
　本クローンを１００μｇ／ｍＬのカナマイシン、３４μｇ／ｍＬのクロラムフェニコール、１００μＭ／ｍＬのＩＰＴＧを含むＬＢ液体培地で、３７℃で終夜培養した。菌体を回収したのち、５０ｍＭのリン酸緩衝液(ｐＨ７.５)で希釈したBugBuster Protein Extraction Reagentを用いて可溶性タンパクを抽出し、Bradford法により抽出されたタンパク質を定量した。得られた一定量の菌体抽出タンパク質に対し、終濃度において０.５ｍＭのカテコールを添加し、マイクロプレートリーダー分光光度計により３７５ｎｍに最大吸収波長を持つ分解産物(２－ヒドロキシムコネート　セミアルデヒド)を定量した。その結果、反復配列の繰り返し数により、カテコール２，３－ジオキシゲナーゼによるカテコール分解産物の生成量が異なることを明らかとした(図６)。
　PET-EDO-N0、PET-EDO-N1については、可溶性のカテコール２，３－ジオキシゲナーゼを生産せず、不溶性タンパクとして生産した。実際に、PET-EDO-N0について、可溶性タンパクを生産させるためには、培養温度、培地、ＩＰＴＧ濃度を厳密に調整する必要がある(参考文献：FEMS Microbiology Ecology,69, 472-480, 2009)。ここで、カテコール２，３－ジオキシゲナーゼの上流に当該配列を３以上付加したプラスミドをもつ大腸菌においては、特段の工夫をすることなく通常の大腸菌の生育条件において可溶性のカテコール２，３－ジオキシゲナーゼの生産が見られた。 (Example 3: E. coli BL21 (DE3) strain host, pET28a vector, catechol 2,3-dioxygenase gene production difference in 2-hydroxymuconate semialdehyde production)
First, the E. coli mutant strains PUC-EDO-N0, PUC-EDO-N1, PUC-EDO-N2, PUC-EDO-N3, PUC-EDO-N4, PUC-EDO-N5, and PUC-EDO used in Example 2 were used. -N6 and PUC-EDO-N7 were cultured at 37 ° C. in LB medium containing 100 μg / mL ampicillin. By extracting the plasmid from the cell culture medium and digesting with XbaI and HindIII, only the region containing the repetitive sequence and the catechol 2,3-dioxygenase gene was excised, and the ligase reaction with the pET28a vector also digested with XbaI and HindIII Went. DNA was recovered and introduced into E. coli BL21 (DE3) strain by the heat shock method, and LB containing 100 μg / mL kanamycin, 34 μg / mL chloramphenicol, 100 μM / mL IPTG, 40 μg / mL X-gal A transformant was obtained by culturing overnight at 37 ° C. on an agar medium. As a result of spraying catechol, colonies showing a yellow color were selected and the nucleotide sequence was determined. The obtained clones were PET-EDO-N0, PET-EDO-N1 (tgacatgct; the repetitive sequence actually used; the same applies hereinafter), PET-EDO-N2 (tgacatgattgacatgct (SEQ ID NO: 50)), PET- EDO-N3 (taacatgattgacatgattgacatgct (SEQ ID NO: 51)), PET-EDO-N4 (taacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 52)), PET-EDO-N5 (tgacatgattaacatgattaacatgattgacatgattgacatgct (ET-49EDt) 53)) and PET-EDO-N7 (tgacatgattgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 54)). On the plasmids possessed by these strains, the repetitive sequence is inserted 12 bp upstream of the target gene. In addition, T7 promoter and lac operator are originally present on the pET28a vector and are located upstream of the repetitive sequence.
This clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 100 μM / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified. A final amount of 0.5 mM catechol is added to the obtained microbial extract protein, and a degradation product (2-hydroxymuconate semialdehyde) having a maximum absorption wavelength at 375 nm by a microplate reader spectrophotometer Was quantified. As a result, it was clarified that the amount of catechol degradation products produced by catechol 2,3-dioxygenase varies depending on the number of repeated sequences (FIG. 6).
PET-EDO-N0 and PET-EDO-N1 did not produce soluble catechol 2,3-dioxygenase, but were produced as insoluble proteins. Actually, in order to produce soluble protein for PET-EDO-N0, it is necessary to strictly adjust the culture temperature, medium, and IPTG concentration (reference: FEMS Microbiology Ecology, 69, 472-480, 2009) . Here, in Escherichia coli having a plasmid in which three or more of the sequences are added upstream of catechol 2,3-dioxygenase, catechol 2,3-dioxygenase soluble under normal growth conditions of E. coli without special measures. Production was seen.
（実施例４：大腸菌JM109株宿主、pCB192ベクター、ベータガラクトシダーゼ遺伝子使用時におけるo-Nitrophenolの生産量の相違）
　はじめに、プロモーター評価用のプラスミドベクターpCB192(国立遺伝学研究所)のベータガラクトシダーゼ遺伝子の上流に、異なる繰り返し数の当該反復配列を挿入する実験を行った。pCB192を鋳型とし、KOD Plus Mutagenesis Kit(TOYOBO社)の説明に従い実験を行った。配列番号３５～４８に記載されたプライマーを使用して、反復配列の繰り返し数が１から６のクローンを作成した。得られたクローンを、PCB-GAL-N1；実際に使用した反復配列を示す。以下同様。）、PCB-GAL-N2（tgacatgattgacatgct (配列番号５０)）、PCB-GAL-N3（taacatgattgacatgattgacatgct (配列番号５１)）、PCB-GAL-N4（taacatgattaacatgattgacatgattgacatgct (配列番号５２)）、PCB-GAL-N5（tgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号４９)）、PCB-GAL-N6（tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号５３)）と名付けた。これらの株が保有するプラスミド上においては、目的遺伝子の40bp上流に当該反復配列が挿入されている。また、pCB192ベクター上にはpromoterおよびoperator配列は存在しない。
　本クローンを１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧを含むＬＢ液体培地で、３７℃で終夜培養した。菌体を回収したのち、５０ｍＭのリン酸緩衝液(ｐＨ７.５)で希釈したBugBuster Protein Extraction Reagentを用いて可溶性タンパクを抽出し、Bradford法により抽出されたタンパク質を定量した。得られた一定量の菌体抽出タンパク質に対し、終濃度において３４ｍＭのONPG(o-Nitrophenyl-β-D-galactopyranoside)を添加し、マイクロプレートリーダー分光光度計により４１０ｎｍに最大吸収波長を持つ分解産物ONP(o-Nitrophenol)を定量した。その結果、反復配列の繰り返し数により、ONPの生成量が異なることを明らかとした(図７)。 (Example 4: Difference in production amount of o-Nitrophenol when using E. coli JM109 host, pCB192 vector, beta-galactosidase gene)
First, an experiment was conducted in which a different number of repetitive sequences were inserted upstream of the beta galactosidase gene of a plasmid vector pCB192 (National Institute of Genetics) for promoter evaluation. Experiments were performed using pCB192 as a template according to the description of KOD Plus Mutagenesis Kit (TOYOBO). Using the primers described in SEQ ID NOs: 35 to 48, clones having 1 to 6 repeats were prepared. The obtained clone is PCB-GAL-N1; the repetitive sequence actually used is shown. The same applies hereinafter. ), PCB-GAL-N2 (tgacatgattgacatgct (SEQ ID NO: 50)), PCB-GAL-N3 (taacatgattgacatgattgacatgct (SEQ ID NO: 51)), PCB-GAL-N4 (taacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 52)), PCB-GAL-N5 tgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 49)) and PCB-GAL-N6 (tgacatgcttgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 53)). On the plasmids possessed by these strains, the repetitive sequence is inserted 40 bp upstream of the target gene. In addition, promoter and operator sequences do not exist on the pCB192 vector.
This clone was cultured overnight at 37 ° C. in an LB liquid medium containing 100 μg / mL ampicillin and 100 μM / mL IPTG. After recovering the cells, soluble proteins were extracted using BugBuster Protein Extraction Reagent diluted with 50 mM phosphate buffer (pH 7.5), and the proteins extracted by the Bradford method were quantified. Degradation product having a maximum absorption wavelength at 410 nm with a microplate reader spectrophotometer by adding 34 mM ONPG (o-Nitrophenyl-β-D-galactopyranoside) at a final concentration to the obtained constant amount of cell-derived protein. ONP (o-Nitrophenol) was quantified. As a result, it was clarified that the amount of ONP produced differs depending on the number of repeats of the repeat sequence (FIG. 7).
（実施例５：Pseudomonas putida KT2440株宿主、pMMB503EHベクター、カテコール２，３－ジオキシゲナーゼ遺伝子使用時における２－ヒドロキシムコネート　セミアルデヒドの生産量の相違）
　まずは、実施例２で使用した大腸菌変異株PUC-EDO-N1、PUC-EDO-N5を１００μｇ／ｍＬのアンピシリンを含むＬＢ培地で、３７℃で培養した。菌体培養液よりプラスミドを抽出し、XbaIおよびHindIIIで消化することにより、当該反復配列とカテコール２，３－ジオキシゲナーゼ遺伝子を含む領域のみを切り出し、同じくXbaIおよびHindIIIで消化したpMMB503EHベクターとligase反応を行った。ＤＮＡを回収し、ヒートショック法で大腸菌JM109株に導入し、１００μｇ／ｍＬのストレプトマイシンを含むＬＢ寒天培地で、３７℃で終夜培養し、形質転換体を得た。コロニーを無差別に選択し、制限酵素処理をおよび塩基配列を決定することにより、pMMB503のｔａｃプロモーター下に、当該反復配列(繰り返し数１および５)とカテコール２，３－ジオキシゲナーゼ遺伝子を保有する、EC-PMM-EDO-N1（tgacatgct；実際に使用した反復配列を示す。以下同様。）およびEC-PMM-EDO-N5（tgacatgattaacatgattaacatgattgacatgattgacatgct (配列番号４９)）を得た。これらの株が保有するプラスミド上においては、目的遺伝子の12bp上流に当該反復配列が挿入されている。 (Example 5: Pseudomonas putida KT2440 strain host, pMMB503EH vector, difference in production of 2-hydroxymuconate semialdehyde when using catechol 2,3-dioxygenase gene)
First, the E. coli mutant strains PUC-EDO-N1 and PUC-EDO-N5 used in Example 2 were cultured at 37 ° C. in an LB medium containing 100 μg / mL ampicillin. By extracting the plasmid from the cell culture medium and digesting with XbaI and HindIII, only the region containing the repetitive sequence and the catechol 2,3-dioxygenase gene was excised, and the ligase reaction with the pMMB503EH vector also digested with XbaI and HindIII Went. DNA was collected, introduced into E. coli strain JM109 by the heat shock method, and cultured overnight at 37 ° C. on an LB agar medium containing 100 μg / mL streptomycin to obtain a transformant. By selecting colonies indiscriminately, performing restriction enzyme treatment and determining the base sequence, the repetitive sequences (repeat numbers 1 and 5) and the catechol 2,3-dioxygenase gene are retained under the pacB503 tac promoter. EC-PMM-EDO-N1 (tgacatgct; the actually used repetitive sequence is shown below) and EC-PMM-EDO-N5 (tgacatgattaacatgattaacatgattgacatgattgacatgct (SEQ ID NO: 49)) were obtained. On the plasmids possessed by these strains, the repetitive sequence is inserted 12 bp upstream of the target gene.
　まず、宿主となるPseudomonas putida KT2440株についてコンピテントセルを作製した。KT2440株を、１００μｇ／ｍＬのアンピシリンを含むＬＢ液体培地で１２時間培養した。培養液の一部をＬＢ液体培地において４時間再培養した後、菌体を回収し、冷却滅菌水に懸濁した。再び遠心分離によって菌体を回収したのち、冷却滅菌水に懸濁した。さらに遠心分離によって回収した菌体に、少量のグリセロールを加え、懸濁したのちに－８０℃で急速に冷凍し、使用時まで保管した。つぎに、EC-PMM-EDO-N1およびEC-PMM-EDO-N5クローンを、１００μｇ／ｍＬのストレプトマイシンを含むＬＢ液体培地で、３７℃で培養し、菌体培養液よりプラスミドを抽出した。プラスミドPMM-EDO-N1およびPMM-EDO-N5をエレクトロポレーション法(BIO-RAD社、MicroPulser)により、Pseudomonas putida KT2440株に導入した。得られた形質展開体をPS-PMM-EDO-N1およびPS-PMM-EDO-N5と名付けた。
　本クローンを１００μｇ／ｍＬのストレプトマイシンと１００μｇ／ｍＬのアンピシリン、１００μＭ／ｍＬのＩＰＴＧを含むＬＢ液体培地で、３０℃で終夜培養した。菌体を回収したのち、５０ｍＭのリン酸緩衝液(ｐＨ７.５)出希釈したBugBuster Protein Extraction Reagent (Merck社)を用いて可溶性タンパクを抽出し、Bradford法により抽出されたタンパク質を定量した。得られた一定量の菌体抽出タンパク質に対し、終濃度において０.５ｍＭのカテコールを添加し、マイクロプレートリーダー分光光度計(TECAN社)により３７５ｎｍに最大吸収波長を持つ分解産物(２－ヒドロキシムコネート　セミアルデヒド)を定量した。その結果、反復配列の繰り返し数により、カテコール２，３－ジオキシゲナーゼによるカテコール分解産物の生成量が異なることを明らかとした(図８)。 First, competent cells were prepared for the Pseudomonas putida KT2440 strain serving as a host. The KT2440 strain was cultured for 12 hours in an LB liquid medium containing 100 μg / mL ampicillin. A part of the culture solution was re-cultured in LB liquid medium for 4 hours, and then the cells were collected and suspended in cold sterilized water. The cells were collected again by centrifugation and then suspended in chilled sterilized water. Further, a small amount of glycerol was added to the cells recovered by centrifugation, suspended, and then rapidly frozen at −80 ° C. and stored until use. Next, EC-PMM-EDO-N1 and EC-PMM-EDO-N5 clones were cultured at 37 ° C. in an LB liquid medium containing 100 μg / mL streptomycin, and a plasmid was extracted from the cell culture medium. Plasmids PMM-EDO-N1 and PMM-EDO-N5 were introduced into Pseudomonas putida KT2440 strain by electroporation (BIO-RAD, MicroPulser). The obtained transformants were named PS-PMM-EDO-N1 and PS-PMM-EDO-N5.
This clone was cultured overnight at 30 ° C. in an LB liquid medium containing 100 μg / mL streptomycin, 100 μg / mL ampicillin and 100 μM / mL IPTG. After recovering the cells, soluble protein was extracted using BugBuster Protein Extraction Reagent (Merck) diluted and diluted with 50 mM phosphate buffer (pH 7.5), and the protein extracted by the Bradford method was quantified. 0.5 mM catechol at a final concentration is added to a certain amount of the cell-derived protein obtained, and a degradation product (2-hydroxymuco) having a maximum absorption wavelength at 375 nm by a microplate reader spectrophotometer (TECAN). Nate semialdehyde). As a result, it was clarified that the amount of catechol degradation products produced by catechol 2,3-dioxygenase varies depending on the number of repeated sequences (FIG. 8).
（実施例６：酵母Saccharomyces cerevisiae YPH500株宿主、pCLYベクター、ルシフェラーゼ遺伝子使用時におけるルシフェリンの生産量の相違）
　分泌型ルシフェラーゼ遺伝子（CLuc）を含み、プロモーターをその上流に含まないレポータープラスミド（pCLY-MCS、アトー株式会社）を用いて、CYC1最小プロモーター（出芽酵母CYC1遺伝子由来）または、反復配列をCYC1最小プロモーターの5’-末端に付与した各種プロモーターを、CLuc遺伝子の上流に導入したレポータープラスミドを以下の方法で作製した。
　pCLY-MCSを鋳型として、inv_pCLY-MCS_R01およびinv_pCLY-MCS_F01をプライマーとして用いて、Platinum SuperFi DNA polymerase（Thermo Fisher Scientific）により、プレ変性９６℃、２分の後、変性９６℃、１０秒、アニーリング５５℃、１０秒、伸張６８℃、４分のサイクルを３５サイクルの後、最終伸張６８℃、５分の条件でＰＣＲ反応を行なった。その後、アガロース電気泳動による精製によって、目的のpCLY-MCSベクターフラグメント（７，２６５ｂｐ）を得た。 (Example 6: Yeast Saccharomyces cerevisiae YPH500 strain host, pCLY vector, difference in luciferin production when using luciferase gene)
Using a reporter plasmid (pCLY-MCS, ATTO Co., Ltd.) that contains a secreted luciferase gene (CLuc) and does not contain a promoter upstream, a CYC1 minimal promoter (derived from the budding yeast CYC1 gene) or a repetitive sequence is a CYC1 minimal promoter A reporter plasmid in which various promoters added to the 5′-end of the gene were introduced upstream of the CLuc gene was prepared by the following method.
Using pCLY-MCS as a template and inv_pCLY-MCS_R01 and inv_pCLY-MCS_F01 as primers, pre-denaturation at 96 ° C. for 2 minutes, followed by denaturation at 96 ° C. for 10 seconds, annealing 55 The PCR reaction was performed under the conditions of a final extension of 68 ° C. for 5 minutes after 35 cycles of 10 ° C., extension 68 ° C. and 4 minutes. Then, the target pCLY-MCS vector fragment (7,265 bp) was obtained by purification by agarose electrophoresis.
　また、各種プロモーターフラグメントの調製は、出芽酵母Saccharomyces cerevisiae S288C由来ゲノムＤＮＡを鋳型として、if_PCYC1min_F01およびif_PCYC1min_R01プライマーセット、if_N1_PCYC1min_F01およびif_PCYC1min_R01プライマーセット、if_N2_PCYC1min_F01およびif_PCYC1min_R01プライマーセット、if_N3_PCYC1min_F01およびif_PCYC1min_R01プライマーセットをそれぞれ用いて、Platinum SuperFi DNA polymeraseにより、プレ変性９６℃，２分の後、変性９６℃，１０秒、アニーリング５５℃，１０秒、伸張６８℃，３０秒のサイクルを３５サイクルの後、最終伸張６８℃，５分の条件でＰＣＲ反応を行なった。その後、アガロースゲル電気泳動による精製によって、それぞれPCYC1minプロモーターフラグメント（１７３ｂｐ）、N1-PCYC1minプロモーターフラグメント（１８２ｂｐ）、N2-PCYC1minプロモーターフラグメント（１９１ｂｐ）、およびN3-PCYC1minプロモーターフラグメント（２００ｂｐ）を得た。
　得られたpCLY-MCSベクターフラグメントおよび、各プロモーターフラグメント(PCYC1min、N1-PCYC1min、N2-PCYC1min、およびN3-PCYC1minプロモーターフラグメント)をそれぞれ用い、In-Fusion HD Cloning Kit(タカラバイオ)を用いて、製品マニュアルに基づきIn-Fusion反応を行なった。本In-Fusion反応液を用いて、大腸菌DH5α株(Competent Quick DH5α、東洋紡)の形質転換を行い、各レポータープラスミドを有する形質転換体を得た。得られた形質転換体より、Monarch Plasmid Miniprep Kit(New England Biolabs)を用いてプラスミドを精製し、サンガーシークエンシング（ユーロフィンジェノミクス）による塩基配列の決定によって、各レポータープラスミドpCLY-PCYC1min, pCLY-N1-PCYC1min(tgacatgct；実際に使用した反復配列を示す。以下同様。), pCLY-N2-PCYC1min(tgacatgattgacatgct (配列番号５０)),およびpCLY-N3-PCYC1min(taacatgattgacatgattgacatgct (配列番号５１))を作製した。 In addition, various promoter fragments were prepared using the budding yeast Saccharomyces cerevisiae S288C-derived genomic DNA as a template, if_PCYC1min_F01 and if_PCYC1min_R01 primer set, if_N1_PCYC1min_F01 and if_PCYC1min_R01 primer set, if_N2_PC1if1min_C01 With SuperFi DNA polymerase, pre-denaturation 96 ° C., 2 minutes, denaturation 96 ° C., 10 seconds, annealing 55 ° C., 10 seconds, extension 68 ° C., 30 seconds after 35 cycles, final extension 68 ° C., 5 minutes PCR reaction was performed under the conditions of Thereafter, PCYC1min promoter fragment (173 bp), N1-PCYC1min promoter fragment (182 bp), N2-PCYC1min promoter fragment (191 bp), and N3-PCYC1min promoter fragment (200 bp) were obtained by purification by agarose gel electrophoresis, respectively.
Using the obtained pCLY-MCS vector fragment and each promoter fragment (PCYC1min, N1-PCYC1min, N2-PCYC1min, and N3-PCYC1min promoter fragments), using In-Fusion HD Cloning Kit (Takara Bio) In-Fusion reaction was performed based on the manual. Using this In-Fusion reaction solution, E. coli DH5α strain (Competent Quick DH5α, Toyobo) was transformed to obtain transformants having each reporter plasmid. Plasmids were purified from the resulting transformants using the Monarch Plasmid Miniprep Kit (New England Biolabs), and each reporter plasmid pCLY-PCYC1min, pCLY-N1 was determined by determining the nucleotide sequence by Sanger sequencing (Eurofin Genomics). -PCYC1min (tgacatgct; shows the repetitive sequence actually used; the same applies below), pCLY-N2-PCYC1min (tgacatgattgacatgct (SEQ ID NO: 50)), and pCLY-N3-PCYC1min (taacatgattgacatgattgacatgct (SEQ ID NO: 51)) .
　作製した各レポータープラスミドを用いて、宿主酵母株としてYPH500株の形質転換を行なった。形質転換は、Frozen-EZ Yeast Transformation II Kit (Zymo Research)を用いて、製品マニュアルに基づき行なった。形質転換後の細胞は、平板合成培地（SC-ura平板培地: 6.7g/L Yeast Nitrogen Base without Amino Acids [Difco]、1.92g/L Yeast Synthetic Drop-out Media Supplements without uracil [Sigma-Aldrich]、2(w/v)% D -Glucose [Sigma-Aldrich]、2(w/v)% Bacto Agar [Difco]）に植菌し、３０℃にて３日間培養を行なった。得られた各形質転換体(３コロニーずつ)をレポーターアッセイ実験に用いた。
上記で作製した各形質転換体は、液体合成培地(SC-ura+PPB 液体培地)を用いて培養を行なった。本液体培地の組成は、6.7g/L Yeast Nitrogen Base without Amino Acids (Difco)、1.92g/L Yeast Synthetic Drop-out Media Supplements without uracil (Sigma-Aldrich)、0.2M Potassium phosphate buffer (pH6.0)、2(w/v)% D-Glucose (Sigma-Aldrich)からなる。前培養として、９６穴ディープウェルプレートに１ｍＬの本液体培地を分注し、各形質転換株(３コロニーずつ)を植菌後、３０℃で３日間の振盪培養を行なった。その後、本培養として、同様に１ｍＬの本合成液体培地を分注した９６穴ディープウェルプレートに、１０ｕＬの前培養液を植菌し、３０℃で３日間の振盪培養を行なった。本培養開始後、２４時間ごと(２４、４８、および７２時間目)に、培養液のサンプリングを行い、培地吸光度(OD600)の測定および、培地中に分泌されたCLuc活性の測定を行なった。培地吸光度(OD600)は、９６穴透明マイクロプレートに２００μＬの本培養液を分注し、マイクロプレートリーダー(Infinite 200 PRO[Tecan])を用いて６００ｎｍでの吸光度を測定した。また、CLuc活性測定は、CLucレポーターアッセイキット(アトー株式会社)を用いて、製品マニュアルに基づき行なった。９６穴黒色マイクロプレートに２５μＬの本培養液を分注し、７５μＬのCLuc発光基質溶液を加え、５秒間の発光をマイクロプレートリーダー(Infinite 200 PRO)を用いて測定した。得られたCLuc活性値は、培地吸光度(OD600)で除算することで、菌体量によるノーマライズを行なった。 Using each prepared reporter plasmid, the YPH500 strain was transformed as a host yeast strain. Transformation was performed based on the product manual using Frozen-EZ Yeast Transformation II Kit (Zymo Research). The transformed cells were prepared using a plate synthesis medium (SC-ura plate medium: 6.7 g / L Yeast Nitrogen Base without Amino Acids [Difco], 1.92 g / L Yeast Synthetic Drop-out Media Supplements without uracil [Sigma-Aldrich], 2 (w / v)% D-Glucose [Sigma-Aldrich], 2 (w / v)% Bacto Agar [Difco]) and inoculated at 30 ° C. for 3 days. Each transformant obtained (3 colonies) was used for reporter assay experiments.
Each transformant produced above was cultured using a liquid synthetic medium (SC-ura + PPB liquid medium). The composition of this liquid medium is 6.7 g / L Yeast Nitrogen Base without Amino Acids (Difco), 1.92 g / L Yeast Synthetic Drop-out Media Supplements without uracil (Sigma-Aldrich), 0.2 M Potassium phosphate buffer (pH 6.0) 2 (w / v)% D-Glucose (Sigma-Aldrich). As preculture, 1 mL of this liquid medium was dispensed into a 96-well deep well plate, and after inoculation of each transformed strain (3 colonies), shaking culture was performed at 30 ° C. for 3 days. Thereafter, as the main culture, 10 uL of the preculture solution was inoculated into a 96-well deep well plate in which 1 mL of the main synthetic liquid medium was similarly dispensed, and the shaking culture was performed at 30 ° C. for 3 days. The culture medium was sampled every 24 hours (24, 48, and 72 hours) after the start of the main culture, and the absorbance of the medium (OD600) and the CLuc activity secreted into the medium were measured. As for the medium absorbance (OD600), 200 μL of the main culture solution was dispensed into a 96-well transparent microplate, and the absorbance at 600 nm was measured using a microplate reader (Infinite 200 PRO [Tecan]). The CLuc activity was measured using a CLuc reporter assay kit (Ato Corporation) based on the product manual. 25 μL of the main culture solution was dispensed into a 96-well black microplate, 75 μL of a CLuc luminescent substrate solution was added, and luminescence for 5 seconds was measured using a microplate reader (Infinite 200 PRO). The obtained CLuc activity value was divided by the absorbance of the medium (OD600) to perform normalization according to the amount of cells.
　前述のレポーターアッセイの結果、１回(pCLY-N1-PCYC1min)または２回(pCLY-N2-PCYC1min)の反復配列をCYC1最小プロモーターの5’-末端に付加した場合、CYC1最小プロモーター(pCLY-PCYC1min)を用いた場合の、約４倍のCLuc活性を示した。さらに、３回の反復配列をCYC1最小プロモーターの5’-末端に付加したもの(pCLY-N3-PCYC1min)では、約７倍のCLuc活性を示した(図９)。本結果から、本反復配列は、出芽酵母由来最小プロモーターにおいても、その転写活性の改善に寄与可能であることが示唆された。 As a result of the above-mentioned reporter assay, when a repetitive sequence once (pCLY-N1-PCYC1min) or twice (pCLY-N2-PCYC1min) is added to the 5′-end of the CYC1 minimal promoter, the CYC1 minimal promoter (pCLY-PCYC1min) ), The CLuc activity was about 4-fold. Furthermore, when the repetitive sequence of 3 times was added to the 5'-end of the CYC1 minimal promoter (pCLY-N3-PCYC1min), CLuc activity was about 7 times (Fig. 9). This result suggests that this repetitive sequence can contribute to the improvement of transcriptional activity even in the minimal promoter derived from budding yeast.

Claims (13)

  1. 　目的遺伝子の発現量を調節するための核酸であって、下記(ｉ)または(ｉｉ)に記載の塩基配列からなる核酸：
    （ｉ）５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’
    　(上記塩基配列において、ｎは１～２０の整数である。)、または、
    （ｉｉ）上記(ｉ)に示される塩基配列において１または数個の塩基が置換、付加、または、欠失した塩基配列からなり、かつ、前記塩基配列の下流に位置する遺伝子の発現量を調節することのできる塩基配列。     A nucleic acid for regulating the expression level of a target gene, the nucleic acid comprising the base sequence described in (i) or (ii) below:
    (I) 5 '-[T (G / A) ACATG (A / C) T] n-3'
    (In the above base sequence, n is an integer of 1 to 20), or
    (Ii) Regulating the expression level of a gene consisting of a base sequence in which one or several bases are substituted, added, or deleted in the base sequence shown in (i) above and located downstream of the base sequence A base sequence that can be used.
  2. 　請求項１に記載の核酸であって、
    前記(ｉ)５’－［Ｔ(Ｇ／Ａ)ＡＣＡＴＧ(Ａ／Ｃ)Ｔ］ｎ－３’で示される塩基配列におけるｎが１～７の整数である、核酸。     The nucleic acid according to claim 1, wherein
    (I) a nucleic acid in which n in the base sequence represented by 5 ′-[T (G / A) ACATG (A / C) T] n-3 ′ is an integer of 1 to 7.
  3. 　請求項１または２に記載の核酸を含むベクター。 A vector comprising the nucleic acid according to claim 1 or 2.
  4. 　請求項３に記載のベクターであって、
    　前記ベクターは発現量を調整する目的遺伝子をさらに含み、前記核酸が前記目的遺伝子の発現量を調節可能なように上流に配置されている、ベクター。     A vector according to claim 3,
    The vector further comprises a target gene for adjusting the expression level, and the nucleic acid is arranged upstream so that the expression level of the target gene can be controlled.
  5. 　請求項３または４に記載のベクターを含む宿主細胞。 A host cell comprising the vector according to claim 3 or 4.
  6. 　請求項５に記載のベクターを含む宿主細胞であって、
    　前記目的遺伝子が宿主細胞に対する外来遺伝子である、宿主細胞。     A host cell comprising the vector of claim 5 comprising
    A host cell, wherein the target gene is a foreign gene for the host cell.
  7. 　目的遺伝子を発現させる方法であって、
    　請求項１または２に記載の核酸が上流に連結された前記目的遺伝子を発現させる工程を含む、発現方法。     A method of expressing a target gene,
    An expression method comprising the step of expressing the target gene to which the nucleic acid according to claim 1 or 2 is linked upstream.
  8. 　請求項７に記載の発現方法であって、
    　前記核酸が、前記目的遺伝子の発現量が所望の量となるように選択された反復数を有する塩基配列からなる核酸である、発現方法。     The expression method according to claim 7, wherein
    The expression method, wherein the nucleic acid is a nucleic acid having a base sequence having a number of repetitions selected so that the expression level of the target gene is a desired amount.
  9. 　請求項７または８に記載の発現方法であって、
    　前記目的遺伝子を発現させる工程が、請求項４に記載のベクターに含まれる前記目的遺伝子を細胞系または無細胞系において発現させる工程である、発現方法。     The expression method according to claim 7 or 8, comprising:
    The expression method, wherein the step of expressing the target gene is a step of expressing the target gene contained in the vector according to claim 4 in a cell system or a cell-free system.
  10. 　請求項７または８に記載の目的遺伝子を発現させる方法であって、
    　前記目的遺伝子が細胞内の内在遺伝子であり、
    　前記目的遺伝子を発現させる工程の前に、請求項１または２に記載の核酸を前記細胞の染色体上に存在する前記目的遺伝子の上流に導入する工程をさらに含む、発現方法。     A method for expressing the target gene according to claim 7 or 8,
    The target gene is an endogenous gene in the cell;
    An expression method further comprising the step of introducing the nucleic acid according to claim 1 or 2 upstream of the target gene present on the chromosome of the cell before the step of expressing the target gene.
  11. 　宿主細胞を用いたタンパク質の産生方法であって、
    　前記宿主細胞において前記タンパク質をコードする遺伝子の発現を促し、前記タンパク質を産生させる工程であって、前記宿主細胞は前記タンパク質をコードする遺伝子の上流に請求項１に記載の核酸を有する工程を含む、
    タンパク質の産生方法。     A method for producing a protein using a host cell, comprising:
    The step of promoting the expression of a gene encoding the protein in the host cell and producing the protein, wherein the host cell comprises the step of having the nucleic acid according to claim 1 upstream of the gene encoding the protein. ,
    Protein production method.
  12. 　請求項１１に記載のタンパク質の産生方法であって、
    　前記タンパク質を産生される工程において、前記宿主細胞が請求項３に記載のベクターを含むものである、
    タンパク質の産生方法。     A method for producing the protein according to claim 11, comprising:
    In the step of producing the protein, the host cell comprises the vector of claim 3.
    Protein production method.
  13. 　請求項１１または１２に記載のタンパク質の産生方法であって、
    　前記核酸が、前記目的遺伝子の発現量が所望の量となるように、選択された反復数の塩基配列からなる核酸である、タンパク質の産生方法。     A method for producing the protein according to claim 11 or 12,
    A method for producing a protein, wherein the nucleic acid is a nucleic acid comprising a base sequence of a selected number of repeats so that the expression level of the target gene is a desired amount.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002507205A (en) * 1997-06-25 2002-03-05 ブラック，チャールズ・アレン，ジュニア Compositions and methods for activating a gene of interest

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002507205A (en) * 1997-06-25 2002-03-05 ブラック，チャールズ・アレン，ジュニア Compositions and methods for activating a gene of interest

Non-Patent Citations (2)

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