WO2012043755A1 - シス作用エレメント及びその利用 - Google Patents

シス作用エレメント及びその利用 Download PDF

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WO2012043755A1
WO2012043755A1 PCT/JP2011/072450 JP2011072450W WO2012043755A1 WO 2012043755 A1 WO2012043755 A1 WO 2012043755A1 JP 2011072450 W JP2011072450 W JP 2011072450W WO 2012043755 A1 WO2012043755 A1 WO 2012043755A1
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cis
gene
acting element
region
transformant
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弘明 小石原
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Toyota Motor Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention provides a cis-acting element that positively regulates the expression of a desired gene during protein production using a filamentous fungus, a nucleic acid construct having the cis-acting element, an expression vector, a transformed cell, and the cis-acting element.
  • the present invention relates to a method for producing a used substance.
  • Filamentous fungi belonging to the genus Aspergillus or Trichoderma are known as microorganisms used for the production of various fermented foods, production of substances such as pharmaceuticals (fermentation industry), and the like.
  • filamentous fungi Penicillium spp and Cephalosporium spp are known to produce antibiotics.
  • Trichoderma spp. Is known as a fungus that produces cellulase
  • Aspergillus spp. Is known as a fungus that produces protease and lactase.
  • enzymes such as cellulase and protease are gene products, and therefore productivity can be directly improved by improving the expression level of the gene.
  • productivity in order to improve the productivity of proteins such as the enzymes described above, it is desired to develop means for improving the expression level of a predetermined gene in the filamentous fungus.
  • Non-Patent Document 1 discloses a method for improving the expression of a foreign gene in Trichoderma reesei.
  • the cellobiohydrolase gene (cbh1) promoter is deleted from the region containing the glucose repressor binding site, and the 200 bp region containing the CCAAT box and the Ace2 binding site is repeated.
  • a modified promoter modified so as to be linked is prepared.
  • Non-Patent Document 1 a transformed Trichoderma reesei in which a reporter gene is linked downstream of this modified promoter is prepared, and the resulting transformant is cultured in a lactose-containing medium, and the activity of the modified promoter is determined by a reporter assay. Evaluating. As a result, a promoter obtained by repeating the 200 bp region four times improved the promoter activity by about 1.4 times compared to a promoter having only one region. In addition, the activity of the promoter which repeated the said area
  • Non-Patent Document 1 provides means for enhancing the amount of foreign genes in Trichoderma reesei up to about 1.4 times.
  • better productivity is required, and the expression level of the target gene is required to be further greatly improved.
  • it is required to improve the expression level of genes as described above and to keep production costs low. Acta. Biochim. Biophys. Sin. (2008): 158-165
  • the present invention provides a novel cis-acting element that can significantly improve the expression level of a desired gene when a filamentous fungus is used as a host cell, a nucleic acid construct having the cis-acting element, an expression vector, and transformation. It aims at providing the manufacturing method of the substance using a cell and the said cis-acting element.
  • the present invention includes the following.
  • a cis-acting element having a region in which an XlnR / Ace2 binding sequence (ggctaa) and a Hap complex binding sequence (ccaat) are arranged via a spacer sequence of 0 to 100.
  • the cis-acting element according to (1) comprising a base sequence of nnnggctaannnnnccaatnnnnnn (n is an arbitrary base selected from adenine, cytosine, guanine and thymine: SEQ ID NO: 3).
  • a nucleic acid construct comprising the cis-acting element according to any one of (1) to (4) and a promoter region.
  • An expression vector comprising the cis-acting element according to any one of (1) to (4) and a promoter region located downstream of the cis-acting element.
  • (11) A method for producing a substance, wherein the transformant according to any one of (8) to (10) is cultured, and the target substance is recovered from the cultured medium and / or the transformant.
  • the cis-acting element according to the present invention can greatly improve the expression level of a gene located downstream.
  • the endogenous gene can be highly expressed.
  • a gene incorporated in the expression vector can be highly expressed in a filamentous fungus.
  • the method for producing a substance according to the present invention by using the cis-acting element, the expression level of a predetermined gene is improved, whereby excellent productivity can be achieved. That is, the method for producing a substance according to the present invention can greatly improve the productivity of a protein encoded by a gene whose expression is promoted by the cis-acting element and / or various substances related to the protein.
  • the cis-acting element according to the present invention includes a region having a predetermined base sequence, and has a function of promoting transcription from a promoter region in a gene located downstream.
  • the cis-acting element according to the present invention includes a region in which an XlnR / Ace2 binding sequence (ggctaa) and a Hap complex binding sequence (ccaat) are arranged via a nucleic acid (spacer region) having 0 to 100 bases.
  • the cis-acting element according to the present invention includes a region denoted as 5′-ggctaaN m ccaat-3 ′ (SEQ ID NO: 1) or 5′-ccctatN m ggctaa-3 ′ (SEQ ID NO: 2). That is, the cis-acting element according to the present invention may have an XlnR / Ace2 binding sequence (ggctaa) and a Hap complex binding sequence (ccaat) in this order from the 5 ′ side, or a Hap complex from the 5 ′ side. A body binding sequence (ccaat) and an XlnR / Ace2 binding sequence (ggctaa) may be included in this order.
  • N is an arbitrary base selected from adenine, cytosine, guanine and thymine.
  • m is an integer from 0 to 100. That is, in the above cis-acting element, N m has a base length of 0 to 100 and is composed of an arbitrary base sequence.
  • the length of N m is not particularly limited, but can be, for example, 1 to 100 bases, preferably 1 to 50 bases, more preferably 1 to 20 bases, and 3 to 10 bases. Is most preferable.
  • the case where m is 0 means a case where the XlnR / Ace2 binding sequence (ggctaa) and the Hap complex binding sequence (ccaat) are directly linked without a spacer region.
  • the cis-acting element according to the present invention is a region in which an XlnR / Ace2 binding sequence (ggctaa) and a Hap complex binding sequence (ccaat) are directly linked, or via a nucleic acid (spacer region) of 1 to 100 bases. In other words, it can be said to include the arranged region.
  • the cis-acting element according to the present invention preferably has a structure in which a plurality of regions including the above-described XlnR / Ace2 binding sequence (ggctaa), Hap complex binding sequence (ccaat) and spacer region are repeatedly arranged.
  • arranging a plurality of times repeatedly means arranging the regions in tandem via a linker sequence having a predetermined base length.
  • the linker sequence means a region having a predetermined base length arranged between a pair of adjacent regions.
  • the base length of the linker sequence is not particularly limited, it may be a base length of 1 to 100 in the same manner as described above N m.
  • a configuration including a region consisting of nnnggctaannnnnnccaatnnnnnnn 5 ′ side ⁇ 3 ′ side: SEQ ID NO: 3)
  • n is selected from adenine, cytosine, guanine and thymine. Any base).
  • SEQ ID NO: 3 6 bases sandwiched between ggctaa and ccaat are spacer regions, and 3 bases on the 3 ′ side and 6 bases on the 5 ′ side mean linker sequences.
  • examples of the cis-acting element according to the present invention include a region consisting of ttaggctaaacgtacccaatgataag (SEQ ID NO: 4).
  • SEQ ID NO: 4 ttaggctaaacgtacccaatgataag
  • 6 bases sandwiched between ggctaa and ccaat are spacer regions, and 3 bases on the 3 ′ side and 6 bases on the 5 ′ side mean linker sequences.
  • the number of the regions is limited. However, it may be 1 to 50, for example, preferably 2 to 30, and more preferably 6 to 24.
  • the number of the regions is less than the above range, the effect of improving the transfer activity may not be sufficiently exhibited. Further, the transfer activity is further improved as the number of the regions is increased. However, when the number of the regions exceeds the range, there is a possibility that the transfer activity cannot be further improved.
  • the cis-acting element according to the present invention can improve transcription activity from a promoter arranged downstream by arranging one or a plurality of the above-described regions.
  • downstream means the transcription direction, that is, the direction from the 5 ′ side to the 3 ′ side in the sense strand.
  • a nucleic acid construct having an expression control region excellent in transcriptional activity can be provided.
  • the transcription activity improvement effect by a cis-acting element can be evaluated by linking a reporter gene to the nucleic acid construct and detecting the expression of the reporter gene.
  • the reporter gene is not limited at all, and for example, a luciferase (LUC) gene or a ⁇ -glucuronidase (GUS) gene can be used. Assays using these reporter genes can also be used by appropriately modifying conventionally known protocols.
  • the nucleic acid construct means a nucleic acid including a cis-acting element having one or more of the above-described regions and a promoter region arranged downstream of the cis-acting element.
  • This nucleic acid construct can also be constructed so as to have restriction enzyme recognition sequences at both ends, for example.
  • this nucleic acid construct can be incorporated into a conventionally known expression vector, for example. That is, by incorporating the above-described cis-acting element according to the present invention into an expression vector that enables expression of a desired gene, an expression vector that can improve the expression of the gene at the transcription level can be provided.
  • This expression vector can be prepared by incorporating the above-mentioned cis-acting element into any conventionally known expression vector mainly used for transformation of host cells.
  • the expression vector having the cis-acting element described above may be either introduced into the host cell chromosome or held outside the chromosome.
  • the expression vector may be any of a plasmid vector, a cosmid vector, a phage vector and the like.
  • the expression vector can include an enhancer, a selection marker, a replication origin, a multiple cloning site, and the like.
  • the promoter is not particularly limited as long as it can drive gene expression in the host filamentous fungus.
  • the tef1 promoter derived from A. oryzae
  • cbh1 promoter T. reesei origin
  • amyB promoter A. oryzae origin
  • Other promoters that can be used include ADH3 promoter, tpiA promoter, alcA promoter, taaG2 promoter, gpdA promoter, and the like.
  • a recombinant vector can be prepared by incorporating a desired gene into an expression vector having the cis-acting element described above. By transforming a host cell using this recombinant vector, the gene is transcribed at a high level in the host cell.
  • the host cell is not particularly limited, but is preferably a fungus such as a filamentous fungus, and more preferably a filamentous fungus.
  • Filamentous fungi that can be used for the host are not particularly limited, but Aspergillus sp.
  • Examples include Rhizomucor filamentous fungi such as Rhizomucor miehei, Penicillium notatum, Penicillium filamentous fungi such as Penicillium chrysogenum, Rhizopus filamentous fungi such as Rhizopus oryzae, Acremonium cellulolyticus, Humicola grisea, Thermoaseus aurantiacus.
  • Aspergillus filamentous fungi, especially Aspergillus speroryzae and Trichoderma genus filamentous fungi, especially Trichoderma reesei are preferable as the host.
  • various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method, etc. may be used. Can do.
  • the gene to be introduced into the host using a recombinant vector is not particularly limited, and examples include genes encoding various proteins.
  • alkaline protease gene ⁇ -amylase gene, ascorbate oxidase gene, aspartic protease gene, cellobiohydrolase gene, cellulase gene, cutinase gene, endoglucanase gene, glucoamylase, ⁇ -glucosidase gene, glyoxal oxidase gene, laccase gene , Lignin oxidase gene, lignin peroxidase gene, lipase gene, manganese peroxidase gene, 1,2- ⁇ -mannosidase gene, nuclease gene, pectin lyase gene, pectin methylesterase gene, acid phosphatase gene, polygalacturonase gene, xylanase gene, Examples thereof include ⁇ -xylosi
  • cellobiohydrolase gene endoglucanase gene, ⁇ -glucosidase gene, Aspergillus amylase gene, protease gene, glucoamylase gene and the like derived from Trichoderma spp. are preferable.
  • the cis-acting element according to the present invention is not limited to the form that is introduced into the host cell together with the desired gene as described above.
  • the cis-acting element is applicable to a form that is incorporated into the expression control amount region of the endogenous gene of the host cell. can do.
  • a recombinant in which the cis-acting element according to the present invention is incorporated into the expression control amount region of the endogenous gene of the host cell is also referred to as a transformant together with the form introduced into the host cell together with the desired gene.
  • transcription activity from the promoter can be improved.
  • a nucleic acid construct containing the above-mentioned cis-acting element and a promoter upstream of the coding region of the endogenous gene the transcriptional activity of the endogenous gene can be improved.
  • a conventionally known method such as a method using a Ku gene disruption strain or the like can be used.
  • the above-described one or more cis-acting elements or the above-described nucleic acid construct can be inserted by homologous recombination using base sequence information at the position to be inserted.
  • the ku gene is a gene encoding a protein necessary for non-homologous recombination, and examples thereof include ku70 gene and ku80 gene.
  • Aichi Prefectural Institute of Industrial Technology Research Report (7), 90-93, 2008-12 can be referred to.
  • the transformant having a cis-acting element according to the present invention is particularly preferably cultured in a medium containing xylan.
  • this transformant is cultured in a xylan-containing medium, the transcription promoting activity by the cis-acting element is more effectively exhibited.
  • the XlnR gene contained in the transformant is highly expressed by xylan contained in the medium, and the action of the XlnR transcription factor on the XlnR / Ace2 binding sequence (ggctaa) contained in the cis-acting element functions sufficiently. Conceivable.
  • the xylan-containing medium means a medium containing xylan exceeding the detection limit.
  • the concentration of xylan contained in the liquid medium is not particularly limited, but can be, for example, 0.1 to 15% w / v, and preferably 0.5 to 12% w / v. % W / v is more preferable. If the xylan concentration is below the above range, the induction of expression of the XlnR gene in the transformant is not sufficient, and there is a possibility that the transcription promoting activity by the cis-acting element cannot be achieved sufficiently. If the concentration of xylan exceeds the above range, the substrate in the liquid medium absorbs moisture, which may cause a problem of poor culture due to insufficient stirring.
  • examples of the xylan-containing medium include a medium made from herbs such as wheat, rice, or bacus, a medium made from wood, and a medium made from agricultural residue or waste.
  • a wheat bran medium can be mentioned as a representative example of a medium made from herbs.
  • the target substance is manufactured at a very low cost because it does not contain expensive ingredients. Can do.
  • the substance to be produced means both a protein encoded by a gene transcribed to a high level by the cis-acting element and a substance in which the protein is involved.
  • a substance in which a protein is involved means, for example, a metabolite when the protein is involved in a metabolic pathway as an enzyme.
  • a material in which a protein is involved when the protein is a cellulase, a sugar content by a saccharification reaction using cellulose contained in a medium as a substrate can be mentioned.
  • Example 1 In this example, the function of a uniquely designed cis-acting element was confirmed by expression of a reporter gene.
  • AttB4 is added to the 5 'end of the 471 bp region and the attB1 sequence is added to the 3' end of the 471 bp region.
  • Gene amplification was performed using a pair of primers; A1 and A2.
  • the obtained amplified fragment was subjected to BP reaction with pDONRP4-P1R to prepare an entry clone (pENTR-Ptef1).
  • A1 5'-ggggacaactttgtatagaaaagttgtttctagatagcgagagtaaa-3 '(SEQ ID NO: 5)
  • A2 5'-ggggactgcttttttgtacaaacttggtttgaaggtggtgcgaacttttg-3 '(SEQ ID NO: 6)
  • a cis-acting element (ttaggctaaacgtacccaatgataag: (SEQ ID NO: 4), 26 bp) containing an enhancer region containing ggctaa and a ccaat gene expression regulatory region is obtained.
  • a nucleic acid fragment consisting of a base sequence having SpeI on the 5 ′ side and XhoI restriction enzyme site on the 3 ′ side was synthesized into a tandem sequence of 12 sets.
  • a nucleic acid fragment containing a tandem sequence in which 6 sets of the above cis-acting elements were repeated was synthesized.
  • nucleic acid fragments were introduced into the EcoRV site of the pMD-simple vector (the one having 12 sets of cis-acting elements is called pMD-i12, and the one having 6 sets of cis-acting elements is called pMD-i6).
  • pUNA source: Kitamoto Laboratory, The University of Tokyo
  • pENTR-PamyB a plasmid pUNA (source: Kitamoto Laboratory, The University of Tokyo) containing the promoter and terminator of the amyB gene derived from Aspergillus oryzae and the nitrate-reducing enzyme gene (niaD) as a template
  • niaD nitrate-reducing enzyme gene
  • a nucleic acid fragment having an attB4 at the 5 ′ end and an attB1 sequence at the 3 ′ end of the amyB promoter site was amplified by PCR using B1 and B2.
  • An entry clone was prepared by performing a BP reaction between the obtained nucleic acid fragment and pDONRP4-P1R (pENTR-PamyB).
  • a nucleic acid fragment containing a translation region of ⁇ -glucuronidase gene by PCR using a plasmid pBI221 (manufactured by Clontech) containing ⁇ -glucuronidase (uidA) as a template and a pair of primers; C1 and C2. was amplified.
  • pENTR-GUS plasmid pBI221
  • uidA ⁇ -glucuronidase
  • B1 5'-ggggacaactttgtatagaaaagttgttccagtgaattcatggtgttttg-3 '(SEQ ID NO: 7)
  • B2 5'-ggggactgctttttgtacaaacttggaaatgccttctgtggggtttatt-3 '(SEQ ID NO: 8)
  • C1 5'-atgttacgtcctgtagaaacc-3 '(SEQ ID NO: 9)
  • pDEST-Ptef1 is obtained by performing LR reaction between various entry clones of pENTR-Ptef1, pENTR-GUS and pENTR-niaD and pEST R4-R3 as shown in FIG. Produced.
  • pDEST-Ptefi6 is obtained by performing LR reaction between pENTR-Ptef1i6, pENTR-GUS and pENTR-niaD entry clones and pEST R4-R3 as shown in FIG. Produced.
  • pDEST-Ptefi12 is obtained by performing LR reaction between various entry clones of pENTR-Ptef1i12, pENTR-GUS and pENTR-niaD and pEST R4-R3 as shown in FIG. Produced.
  • pDEST-PamyB is obtained by performing LR reaction with various entry clones of pENTR-PamyB, pENTR-GUS and pENTR-niaD and pEST R4-R3 as shown in FIG. Produced.
  • Tzapek Docs medium containing nitric acid as a single nitrogen source (0.2% NaNO 3 , 0.1% KH 2 PO 4 , 0.05% KCl, 0.05% MgSO 4 .7H 2 O, 2% glucose. , PH 5.5) was used as an indicator, and an individual capable of growing in a Czapek Dox medium containing nitric acid as a single nitrogen source was selected as a transformant. From a plurality of selected transformants, one into which a copy of the transgene (uidA gene) was introduced was selected by genomic Southern analysis using the uidA gene as a probe.
  • Transformants into which one copy of pDEST-Ptef, pDEST-Ptefi12, pDEST-Ptefi6 or pDEST-PamyB plasmid was introduced as described above were named Ptef1, Ptefi12, Ptefi6 and PamyB, respectively.
  • Solid culture was performed by the following method. First, using a 100 ml flask, the seed medium (corn starch 5.6 g, polypeptone 1.8 g, KH 2 PO 4 0.1 g, KCl 0.05 g, MgSO 4 .7H 2 O 0.15 g, CaCl 2 .2H 2 O 0.2 g, distilled water 100 ml) was adjusted to 20 ml, and an appropriate amount of conidia was inoculated and cultured at 30 ° C. and 150 rpm for one day.
  • the seed medium corn starch 5.6 g, polypeptone 1.8 g, KH 2 PO 4 0.1 g, KCl 0.05 g, MgSO 4 .7H 2 O 0.15 g, CaCl 2 .2H 2 O 0.2 g, distilled water 100 ml
  • Liquid culture was performed by the following method. First, the amount of seed medium was 20 ml using a 100 ml flask, an appropriate amount of conidia was inoculated, and cultured at 30 ° C. and 150 rpm for 1 day. Thereafter, 3 ml of the cultured seed medium was added to a liquid culture medium (brass 10 g, ammonium sulfate 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 7H 2 O 0.05 g, distilled water 100 ml / 500 ml flask with baffle), DPY liquid medium (dextrin).
  • a liquid culture medium brass 10 g, ammonium sulfate 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 7H 2 O 0.05 g, distilled water 100 ml / 500 ml flask with baffle
  • DPY liquid medium DPY liquid medium
  • Ptef1i12 improved the GUS activity by 4.92 times than Ptef1, and improved the GUS activity by 2.06 times than PamyB.
  • Ptef1i6 showed a 2.92-fold improvement in GUS activity over Ptef1, and a 1.34-fold improvement in GUS activity over PamyB.
  • Ptef1i12 improved the GUS activity by 3.94 times than Ptef1, and improved the GUS activity by 3.57 times than PamyB.
  • Ptef1i6 improved 2.93 times GUS activity than Ptef1, and 2.65 times GUS activity improved more than PamyB.
  • Ptef1i12 was 1.23 times more GUS activity than Ptef1, but the degree of activity was lower than other culture conditions.
  • the GUS activity in Ptef1i12 was significantly improved even when compared with PamyB, which is generally highly expressed, under conditions in which culture is performed using a bran as a substrate, such as solid culture and liquid culture conditions. It was confirmed that However, liquid culture using lactose did not show much improvement in GUS activity of Ptef1i12. From this result, it became clear that the cis-acting element designed in the present example has a characteristic of further improving gene expression in a medium condition containing xylan such as a fusuma medium.
  • Acta. Biochim. Biophys. Sin. (2008): 158-165 shows that a promoter having a repeat of about 200 bp four times has a promoter activity of about 1.4 times that of a promoter having only one such region. However, it is specified that the activity of the promoter which repeated the region 6 times was almost equivalent to the promoter which repeated the region 4 times.
  • the region of about 200 bp disclosed in Acta. Biochim. Biophys. Sin. (2008): 158-165 is evaluated to be low although the effect of improving gene expression is observed.
  • the effect of improving gene expression is further enhanced by using the approximately 200 bp region disclosed in Acta. Biochim. Biophys. Sin. (2008): 158-165 four times. The effect of improving gene expression does not increase any further.
  • the cis-acting element designed in this example compared with a conventionally known cis-acting element (acta. Biochim. Biophys. Sin. (2008): about 200 bp region disclosed in 158-165). Therefore, the effect of improving gene expression is remarkably excellent.
  • the cis-acting element designed in the present example is larger than the conventionally known cis-acting element (acta. Biochim. Biophys. Sin. (2008): about 200 bp region disclosed in 158-165). Even if these sets are repeatedly used in tandem, the effect of improving gene expression can be enhanced depending on the number of repetitions. For this reason, the cis-acting element designed in this example can adjust the effect of improving gene expression more precisely by appropriately setting the number of repetitions.

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