WO2011099243A1 - Target gene expression method using a specific promoter derived from saccharomyces cerevisiae in kluyveromyces marxianus - Google Patents

Target gene expression method using a specific promoter derived from saccharomyces cerevisiae in kluyveromyces marxianus Download PDF

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WO2011099243A1
WO2011099243A1 PCT/JP2011/000439 JP2011000439W WO2011099243A1 WO 2011099243 A1 WO2011099243 A1 WO 2011099243A1 JP 2011000439 W JP2011000439 W JP 2011000439W WO 2011099243 A1 WO2011099243 A1 WO 2011099243A1
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promoter
target gene
saccharomyces cerevisiae
transformant
kluyveromyces marxianus
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PCT/JP2011/000439
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French (fr)
Japanese (ja)
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倫治 赤田
尚司 星田
政充 井手
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国立大学法人山口大学
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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
    • C12N15/63—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
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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

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  • the present invention relates to a method for expressing a target gene and a method for producing a target gene product. More specifically, the present invention relates to a method for expressing a target gene and a method for producing a target gene product using a specific promoter derived from Saccharomyces cerevisiae in Kluyveromyces marxianus.
  • heterologous protein gene When a heterologous protein gene is expressed in a host, a recombinant polynucleotide in which the heterologous protein gene is operably placed under the control of a promoter that functions in the host is introduced into the host, and the heterologous protein gene is expressed in the transformant.
  • a promoter with high transcription activity is generally used.
  • PGK1 promoter when the host is Saccharomyces cerevisiae, PGK1 promoter, TEF1 promoter, ADH1 promoter, TPI1 promoter, PYK1 promoter, ENO1 promoter, PMA1 promoter, ADH1 promoter, TDH3 promoter, which are high transcription activity promoters derived from Saccharomyces cerevisiae, In some cases, ILV5 promoter, CWP2 promoter and the like are used.
  • Kluyveromyces marcianus is a heat-resistant yeast (for example, Non-Patent Documents 1 and 2).
  • the temperature of the fermentation liquor rises due to the heat of fermentation, so the fermentation broth had to be cooled in order to sustain ethanol fermentation. Therefore, in order to perform ethanol fermentation industrially, a large-scale cooling facility and a large energy cost required for the cooling are indispensable.
  • Kluyveromyces marcianus can grow even at temperatures as high as 48 ° C (Non-Patent Documents 3 and 4), so efficient ethanol fermentation is possible without the need for such cooling equipment and energy costs. It becomes.
  • a promoter exhibits the highest promoter activity in the cell of the species from which the promoter is derived, and in other species, it exhibits no promoter activity itself or a reduced promoter activity.
  • An object of the present invention is to provide a method for expressing a target gene and a method for producing a target gene product using a specific promoter derived from Saccharomyces cerevisiae in Kluyveromyces marxianus.
  • TDH3 promoter from Saccharomyces cerevisiae is known to show no galactose-inducible expression in Saccharomyces cerevisiae, but we use this promoter in Kluyveromyces marxianus. Surprisingly, it was found to show galactose-induced expression.
  • the inventors have completed the present invention based on the above findings.
  • the present invention is characterized in that (1) a promoter selected from the group consisting of TDH3 promoter derived from Saccharomyces cerevisiae, ILV5 promoter, CWP2 promoter, and variants thereof is used in Kluyveromyces marxianus.
  • Transformation obtained by introducing the recombinant polynucleotide described in (A) below or the vector described in (B) below into Kluyveromyces marxianus A method of expressing a target gene as described in (1) above, which comprises a step of culturing the body: (A) a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof Promoters selected from the group and their control A recombinant polynucleotide comprising a target gene operably disposed below: (B) a vector comprising the recombinant polynucleotide described in (A) above; and (3) a TDH3 promoter derived from Saccharomyces cerevisiae as a promoter. And the method for expressing a target gene according to (2) above, wherein the transformant is cultured in the presence of galactose.
  • the present invention is characterized in that (4) a promoter selected from the group consisting of SDH from Saccharomyces cerevisiae, a TDH3 promoter, an ILV5 promoter, a CWP2 promoter, and variants thereof is used in Kluyveromyces marxianus. And (5) a trait obtained by introducing the recombinant polynucleotide described in (A) below or the vector described in (B) below into Kluyveromyces marxianus A step (P) of culturing the transformant, and a step (Q) of recovering the target gene product from the transformant obtained by culturing.
  • a promoter selected from the group consisting of SDH from Saccharomyces cerevisiae, a TDH3 promoter, an ILV5 promoter, a CWP2 promoter, and variants thereof is used in Kluyveromyces marxianus.
  • Production method (A) TDH3 promoter, ILV5 promoter, CWP2 derived from Saccharomyces cerevisiae A recombinant polynucleotide comprising a promoter selected from the group consisting of a lomotor and a mutant thereof, and a target gene operably arranged under the control thereof: (B) the recombination according to (A) above The target gene product according to (5) above, wherein the vector comprises a polynucleotide, and (6) the TDH3 promoter derived from Saccharomyces cerevisiae is used as the promoter, and the transformant is cultured in the presence of galactose.
  • the target gene can be highly expressed. Moreover, according to the method for producing a target gene product of the present invention, the target gene product can be produced with high efficiency.
  • the expression method of the target gene of the present invention is a promoter selected from the group consisting of TDH3 promoter, ILV5 promoter, CWP2 promoter derived from Saccharomyces cerevisiae, and variants thereof (hereinafter summarized) Is also referred to as “promoter in the present invention”) in Kluyveromyces marxianus.
  • promoter in the present invention When the promoter in the present invention is used in Kluyveromyces marxianus, the target gene can be highly expressed.
  • a recombinant polynucleotide described in (A) below or a vector described in (B) below is introduced into Kluyveromyces marxianus. Culturing the resulting transformant.
  • a recombination comprising a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, and a target gene operably arranged under the control thereof
  • Polynucleotide (B) A vector comprising the recombinant polynucleotide according to (A) above:
  • the polynucleotide shown in SEQ ID NO: 1 can be preferably exemplified, and as the ILV5 promoter derived from Saccharomyces cerevisiae, the polynucleotide shown in SEQ ID NO: 2 can be exemplified.
  • Nucleotides can be preferably exemplified, and as the CWP2 promoter derived from Saccharomyces cerevisiae, the polynucleotide shown in SEQ ID NO: 3 can be suitably exemplified.
  • the variant of the promoter in the above (A) means a polynucleotide variant shown in any one of SEQ ID NOs: 1 to 3 and having a promoter activity in Kluyveromyces marxianus. .
  • Such promoter variants include: (A) 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more with respect to the polynucleotide shown in any one of SEQ ID NOs: 1 to 3
  • polynucleotide in which one or two or more nucleotides are deleted, substituted or added is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, Preferably, it means a polynucleotide in which any number of 1 to 5 nucleotides has been deleted, substituted or added.
  • the “stringent condition” in the above (c) means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Specifically, it is 80% or more, preferably 85% or more.
  • the DNAs having the same identity are hybridized with each other and DNAs with lower identity are not hybridized with each other, or a normal Southern hybridization washing condition at 65 ° C., 1 ⁇ SSC solution (1 ⁇ concentration SSC)
  • the composition of the solution can include conditions for hybridization at a salt concentration corresponding to 150 mM sodium chloride, 15 mM sodium citrate), 0.1% SDS, or 0.1 ⁇ SSC, 0.1% SDS. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.
  • Examples of the “polynucleotide hybridizing under stringent conditions” in the above (c) include a polynucleotide having a certain identity or more with a polynucleotide used as a probe, for example, 80% or more, preferably Suitable examples include polynucleotides having 85% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98% or more.
  • Oligonucleotides can be synthesized according to a conventional method using various commercially available DNA synthesizers, for example.
  • the PCR reaction is carried out using a thermal cycler Gene AmpPCRSystem 2400 manufactured by Applied Biosystems, using TaqDNA polymerase (manufactured by Takara Bio Inc.) or KOD-Plus- (manufactured by Toyobo Co., Ltd.). Can be done according to
  • polynucleotide variants can also be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, the polynucleotide shown in any one of SEQ ID NOs: 1 to 3 is contacted with a mutagen agent, irradiated with ultraviolet rays, genetic engineering techniques, etc. By introducing mutations into nucleotides, polynucleotide variants can be obtained. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning 2nd edition, Current Protocols in MolecularlogyBiology, Supplement 1 ⁇ 38, John Wiley and Sons (1987-1997).
  • the promoter in the present invention has promoter activity in Kluyveromyces marxianus. Whether a polynucleotide has promoter activity in Kluyveromyces marxianus is determined by, for example, producing a recombinant polynucleotide in which a reporter gene is operably linked downstream of the polynucleotide, This can be easily confirmed by a well-known reporter assay including a step of transforming Iberomyces marcianus to obtain transformed yeast and a step of measuring the expression level of the reporter gene in the transformed yeast.
  • the secretory luciferase CLuc gene can be operated downstream of the promoter in the present invention.
  • the relative expression level (RLU / OD ⁇ ⁇ l) of the secreted luciferase CLuc in the culture medium when shaken for 48 hours is 10,000 or more, preferably 12,000 or more, more preferably 14,000 or more, more preferably 16000 or more, more Preferably it is preferably 18000 or more.
  • the above-mentioned “high expression” and “high efficiency” indicate that the relative expression level (RLU / ⁇ l) of the secreted luciferase CLuc in the culture medium is 300,000 or more, preferably 450,000 or more, more preferably 60 It is suitably included that it is 10,000 or more, more preferably 750,000 or more.
  • “high expression” in the expression method of the target gene of the present invention and “high efficiency” in the method of producing the target gene product of the present invention described later include a secreted luciferase CLuc downstream of the promoter in the present invention.
  • a transformant obtained by introducing a recombinant polynucleotide operably linked with a gene into Kluyveromyces marxianus was transformed into YPD liquid medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose).
  • the relative expression level (RLU / OD ⁇ ⁇ l) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C. for 48 hours at 37 ° C.
  • the ratio is 3 times or more, preferably 4 times or more, more preferably 5 times the relative expression level (RLU / OD ⁇ ⁇ l) of the secreted luciferase CLuc in the culture medium when shaken at 48 ° C. for 48 hours. More preferably, it includes 7 times or more, more preferably 15 times or more, further preferably 25 times or more, and further preferably 50 times or more.
  • a secretory luciferase is provided downstream of the promoter in the present invention.
  • a transformant obtained by introducing a recombinant polynucleotide operably linked to a CLuc gene into Kluyveromyces marxianus was transformed into a YPGal liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass galactose. ), The relative expression level (RLU / OD ⁇ ⁇ l) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C.
  • the ratio is 3 times or more, preferably 4 times or more relative to the relative expression level (RLU / OD ⁇ ⁇ l) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C. for 48 hours at It is preferably included.
  • a complementary double-stranded polynucleotide can be preferably exemplified, and a complementary double-stranded DNA can be particularly preferably exemplified.
  • target gene operably arranged under the control of the promoter in the present invention of the recombinant polynucleotide (A) above means that the expression of the target gene is caused by binding of a transcription factor to the promoter of the present invention. It means that the promoter of the present invention and its target gene are linked so as to be induced.
  • the “target gene” may be any gene, and a useful protein gene encoding any useful protein can be preferably exemplified.
  • useful protein genes include saccharifying enzyme genes such as cellulase gene and amylase gene, and viral vaccine protein genes.
  • the vector according to the present invention described in (B) above includes the recombinant polynucleotide according to the present invention.
  • Kluyveromyces marcianus can be used to retain the recombinant polynucleotide of the present invention or to transform Vietnameseveromyces marcianus to express the target gene.
  • the vector in the present invention may be linear or circular. Kluyveromyces marxianus can cause recombination at high frequency on a chromosome even if it is a linear vector, and as a result, can be transformed.
  • the vector in the case of a circular vector, if the vector further contains a self-replicating sequence, the vector autonomously replicates in the Kluyveromyces marxianus cell and is retained in the yeast cell. As a result, the vector can be transformed. it can.
  • the vector is not particularly limited as long as the target gene can be expressed in Kluyveromyces marxianus cells.
  • pKD1 can be preferably exemplified. .
  • the transformant in the present invention can be obtained by introducing the recombinant polynucleotide in the present invention or the vector in the present invention into Kluyveromyces marxianus.
  • the transformant in the present invention can highly express the target gene in the cell.
  • the method for introducing the recombinant polynucleotide in the present invention or the vector in the present invention into yeast is not particularly limited, and organisms such as a method using a viral vector, a method using a specific receptor, a cell fusion method, etc.
  • Physical methods such as electroporation method, microinjection method, gene gun method, ultrasonic gene transfer method; chemical methods such as lipofection method, calcium phosphate coprecipitation method, liposome method, DEAE dextran method, etc.
  • Known methods can be exemplified, and among them, the lipofection method can be suitably exemplified in terms of simplicity and high versatility.
  • the recombinant polynucleotide in the present invention or the vector in the present invention has been introduced into the yeast depends on the marker gene as the target gene or in addition to the target gene. It can be easily confirmed by inserting a marker gene into the vector of the invention and confirming the expression of the marker gene in the transformant.
  • the method includes the step of culturing the transformant of the present invention.
  • the “method for culturing the transformant in the present invention” is not particularly limited as long as the transformant can grow.
  • the transformant can be grown under temperature conditions (for example, 25 to 50 ° C., preferably 25 to 40 ° C., more preferably 28 to 33 ° C.) and YPD medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose) for an appropriate time (eg 1 to 10 days, preferably 1 to A method of shaking culture for 5 days, more preferably 1 to 3 days) can be suitably exemplified.
  • the target gene when the TDH3 promoter derived from Saccharomyces cerevisiae is used, the target gene can be expressed more significantly, and therefore it is preferable to use galactose instead of glucose as the sugar source. . That is, among the promoters in the present invention, when the TDH3 promoter derived from Saccharomyces cerevisiae is used, the obtained transformant is preferably cultured in the presence of galactose.
  • the method for producing a target gene product of the present invention is characterized in that the promoter of the present invention is used in Kluyveromyces marxianus.
  • the promoter of the present invention is used in Kluyveromyces marcianus, the target gene product can be produced with high efficiency.
  • a recombinant polynucleotide described in (A) below or a vector described in (B) below is introduced into Kluyveromyces marxianus.
  • a recombination comprising a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, and a target gene operably arranged under the control thereof
  • Polynucleotide (B) A vector comprising the recombinant polynucleotide according to (A) above:
  • the step (P) in the more preferable embodiment of the method for producing the target gene product of the present invention is the same as the step in the more preferable embodiment of the aforementioned target gene expression method of the present invention.
  • the method of “recovering the target gene product from the transformant obtained by culturing” in the above step (Q) is not particularly limited as long as the target gene product can be recovered, and a method using chromatography, A known method such as a method using a tag can be exemplified.
  • Method for increasing the expression efficiency of the target gene of the present invention is characterized in that the promoter of the present invention is used in Kluyveromyces marxianus.
  • the expression efficiency of the target gene can be improved.
  • the same aspect as the preferable aspect in the aforementioned expression method of the target gene of the present invention can be exemplified.
  • TDH3 promoter ScTDH3 promoter
  • ILV5 promoter ScILV5 promoter
  • CWP2 promoter ScCWP2 promoter
  • ScTDH3 promoter expression analysis test 1 (glucose medium)
  • Saccharomyces cerevisiae In order to examine whether the ScTDH3 promoter derived from Saccharomyces cerevisiae can express the target gene in Kluyveromyces marxianus, the following expression analysis test was performed.
  • a comparative transformant 1 (Sc TF ScTDH3p-CLuc) obtained by introducing ScTDH3p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScTDH3 promoter into Saccharomyces cerevisiae.
  • Example transformant 1 (Km TF ScTDH3p-CLuc) obtained by introducing the above ScTDH3p-CLuc into Kluyveromyces marxianus.
  • Both of these transformants were each cultured with shaking at 28 ° C. in a YPD liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass glucose).
  • the culture solution was sampled over time, and the relative expression level (RLU / ⁇ l) of the secreted luciferase CLuc in 1 ⁇ l of the culture solution and the OD600 of the culture solution were measured.
  • Measurement results (relative expression level (RLU / ⁇ l)) immediately after the start of culture, 24 hours after the start of culture, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours ) Is shown in FIG.
  • the graph of FIG. 1 shows the average value of 3 clones for each type of transformant.
  • Example Transformant 1 shows early and remarkable high expression compared to Comparative Example Transformant 1.
  • Example transformant 1 showed high expression immediately after the start of the culture as compared with the Comparative Example transformant 1, and after 48 hours from the start of the culture, the Example transformant 1 Compared to transformant 1, the relative expression level (RLU / ⁇ l) of 139 times or more (relative expression level (RLU / OD ⁇ ⁇ l) of 66 times or more) was shown as a ratio.
  • This “early and remarkable high expression” of Example transformant 1 can be said to be a remarkable effect because it greatly increases the production efficiency per unit time of the target gene product and the like.
  • the TDH3 promoter derived from Saccharomyces cerevisiae exhibits early and remarkable high expression when used in Kluyveromyces marcianus compared to that used in Saccharomyces cerevisiae. It was.
  • a promoter exhibits the highest promoter activity in the cell of the species from which the promoter is derived, and in other species it does not exhibit promoter activity itself or exhibits reduced promoter activity. Considering this, the results of this expression analysis test were unexpected.
  • “activity amount” is synonymous with “relative expression level”.
  • the Example transformant 1 showed a more remarkable high expression.
  • the relative expression level (RLU / ⁇ l) (5.1 times or more) under the condition of galactose medium is 4.9 times or more after 48 hours from the start of the culture as compared with the condition of glucose medium.
  • Fold relative expression level (RLU / OD ⁇ ⁇ l)) and after 120 hours, 4.9-fold relative expression level (RLU / ⁇ l) (4.8-fold relative expression level (RLU / OD)) - ⁇ l)).
  • Example transformant 1 under the galactose medium condition was 833 times or more relative expression level (RLU / ⁇ l) (607 times or more) after 48 hours from the start of the culture, compared with the comparative example transformant 1.
  • Relative expression level (RLU / OD ⁇ ⁇ l)) was shown, and after 120 hours, 86 times or more relative expression level (RLU / ⁇ l) (72 times or more relative expression level (RLU / OD ⁇ ⁇ l)) was shown. .
  • Comparative transformant 2 (Sc TF ScILV5p-CLuc) obtained by introducing ScILV5p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScILV5 promoter into Saccharomyces cerevisiae.
  • Example transformant 2 (Km TF ScILV5p-CLuc) obtained by introducing the above ScILV5p-CLuc into Kluyveromyces marcianus.
  • Comparative transformant 3 (Sc TF ScCWP2p-CLuc) obtained by introducing ScCWP2p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScCWP2 promoter into Saccharomyces cerevisiae.
  • Example transformant 3 (Km TF ScCWP2p-CLuc) obtained by introducing the above ScCWP2p-CLuc into Kluyveromyces marxianus.
  • Example transformant 1 showed a relative expression level (RLU / OD ⁇ ⁇ l) 18 times or more that of Comparative Example transformant 1
  • Example Transformant 2 was The relative expression level (RLU / OD ⁇ ⁇ l) is 7 times or more that of Comparative Example Transformant 2
  • Example Transformant 3 is 4 times or more relative expression compared to Comparative Example Transformant 3.
  • the amount (RLU / OD ⁇ ⁇ l) was indicated.
  • the relative expression level of Example transformant 2 (40882 RLU / OD ⁇ ⁇ l) and the relative expression level of Example transformant 3 (29324 RLU / OD ⁇ ⁇ l) are both shown in Example transformant 1.
  • the ILV5 promoter derived from Saccharomyces cerevisiae (ScILV5 promoter) and the CWP2 promoter (ScCWP2 promoter) are also more prominent when used in Kluyveromyces marxianus than in Saccharomyces cerevisiae. High expression was shown.
  • the present invention can be used particularly effectively in the fields of high expression of a target gene and production of a target gene product with high efficiency.

Abstract

Provided is a method for expressing a target gene. In said method, a specific promoter derived from Saccharomyces cerevisiae (the TDH3 promoter, ILV5 promoter, or CWP2 promoter) is used in Kluyveromyces marxianus. Also provided is a method for manufacturing the product of a target gene.

Description

サッカロマイセス・セレビシエ由来の特定のプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いる目的遺伝子の発現方法Expression method of target gene using specific promoter derived from Saccharomyces cerevisiae in Kluyveromyces marxianus
　本発明は、目的遺伝子の発現方法、及び、目的遺伝子産物の製造方法に関する。より詳細には、サッカロマイセス・セレビシエ（Saccharomyces cerevisiae）由来の特定のプロモーターを、クルイベロマイセス・マルシアヌス（Kluyveromyces marxianus）において用いる、目的遺伝子の発現方法、及び、目的遺伝子産物の製造方法に関する。 The present invention relates to a method for expressing a target gene and a method for producing a target gene product. More specifically, the present invention relates to a method for expressing a target gene and a method for producing a target gene product using a specific promoter derived from Saccharomyces cerevisiae in Kluyveromyces marxianus.
　近年の遺伝子組換え技術の進展とともに、様々な有用なタンパク質の生産を、大腸菌等の微生物を用いて行うことが可能となった。しかし、真核生物由来の異種タンパク質遺伝子を、大腸菌を宿主として発現させると、正常なプロセシングや糖鎖の付加などの翻訳後の正常な修飾が行われないという問題があることが知られている。そのため、真核生物である酵母が、宿主として比較的利用されている。宿主酵母としては、例えば、サッカロマイセス・セレビシエ、ピキア・パストリス、ピキア・メタノリカ、シゾサッカロマイセス・ポンベ、ハンゼヌラ・アノマーラ、クリベロマイセス・ラクティスなどが知られている（例えば特許文献１参照）。 With the recent progress of gene recombination technology, it has become possible to produce various useful proteins using microorganisms such as Escherichia coli. However, it is known that when a heterologous protein gene derived from a eukaryote is expressed in E. coli as a host, normal post-translational modifications such as normal processing and addition of sugar chains are not performed. . Therefore, eukaryotic yeast is relatively utilized as a host. As host yeasts, for example, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, Hansenula anomala, Krivellomyces lactis and the like are known (see, for example, Patent Document 1).
　異種タンパク質遺伝子を宿主にて発現させる際には、宿主内で機能するプロモーターの制御下にその異種タンパク質遺伝子を作動可能に配置した組換えポリヌクレオチドを宿主内に導入し、その形質転換体内においてその異種タンパク質を発現させる。プロモーターの転写活性は、異種タンパク質の発現効率を大きく左右する。そのため、転写活性の高いプロモーターが一般的に用いられている。例えば、宿主がサッカロマイセス・セレビシエである場合は、サッカロマイセス・セレビシエ由来の高転写活性プロモーターであるＰＧＫ１プロモーター、ＴＥＦ１プロモーター、ＡＤＨ１プロモーター、ＴＰＩ１プロモーター、ＰＹＫ１プロモーター、ＥＮＯ１プロモーター、ＰＭＡ１プロモーター、ＡＤＨ１プロモーター、ＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーターなどが用いられることがあった。 When a heterologous protein gene is expressed in a host, a recombinant polynucleotide in which the heterologous protein gene is operably placed under the control of a promoter that functions in the host is introduced into the host, and the heterologous protein gene is expressed in the transformant. Express heterologous proteins. The transcriptional activity of the promoter greatly affects the expression efficiency of the heterologous protein. Therefore, a promoter with high transcription activity is generally used. For example, when the host is Saccharomyces cerevisiae, PGK1 promoter, TEF1 promoter, ADH1 promoter, TPI1 promoter, PYK1 promoter, ENO1 promoter, PMA1 promoter, ADH1 promoter, TDH3 promoter, which are high transcription activity promoters derived from Saccharomyces cerevisiae, In some cases, ILV5 promoter, CWP2 promoter and the like are used.
　ところで、クルイベロマイセス・マルシアヌスは、耐熱性酵母である（例えば、非特許文献１及び２）。通常の酵母でエタノール発酵をする場合、発酵熱によって発酵液の温度が上昇していくため、エタノール発酵を持続させるには発酵液を冷却しなければならなかった。そのため、エタノール発酵を工業的に行うには、大規模な冷却設備と、その冷却に要する多大なエネルギーコストが不可欠となる。しかし、クルイベロマイセス・マルシアヌスは４８℃もの高温でも増殖が可能であるため（非特許文献３及び４）、そのような冷却設備やエネルギーコストを必要とせずに、効率的なエタノール発酵が可能となる。 By the way, Kluyveromyces marcianus is a heat-resistant yeast (for example, Non-Patent Documents 1 and 2). In the case of ethanol fermentation with normal yeast, the temperature of the fermentation liquor rises due to the heat of fermentation, so the fermentation broth had to be cooled in order to sustain ethanol fermentation. Therefore, in order to perform ethanol fermentation industrially, a large-scale cooling facility and a large energy cost required for the cooling are indispensable. However, Kluyveromyces marcianus can grow even at temperatures as high as 48 ° C (Non-Patent Documents 3 and 4), so efficient ethanol fermentation is possible without the need for such cooling equipment and energy costs. It becomes.
　一般的に、プロモーターは、そのプロモーターが由来する生物種の細胞内において最も高いプロモーター活性を示し、他の生物種においては、プロモーター活性そのものを示さないか、あるいは、低下したプロモーター活性を示す。 Generally, a promoter exhibits the highest promoter activity in the cell of the species from which the promoter is derived, and in other species, it exhibits no promoter activity itself or a reduced promoter activity.
特開２００８－２９２３９号公報JP 2008-29239 A
　本発明は、サッカロマイセス・セレビシエ由来の特定のプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いる、目的遺伝子の発現方法、及び、目的遺伝子産物の製造方法を提供することを目的とする。 An object of the present invention is to provide a method for expressing a target gene and a method for producing a target gene product using a specific promoter derived from Saccharomyces cerevisiae in Kluyveromyces marxianus.
　本発明者らは、鋭意研究を行った結果、サッカロマイセス・セレビシエ由来の特定のプロモーター（ＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター）を、クルイベロマイセス・マルシアヌスにおいて用いると、意外なことに、元来の宿主であるサッカロマイセス・セレビシエにおいて用いた場合よりも目的遺伝子が高発現することを見い出した。 As a result of intensive studies, the present inventors have unexpectedly found that when specific promoters derived from Saccharomyces cerevisiae (TDH3 promoter, ILV5 promoter, CWP2 promoter) are used in Kluyveromyces marxianus, It was found that the target gene was expressed at a higher level than that used in Saccharomyces cerevisiae, the host of.
　さらに、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターは、サッカロマイセス・セレビシエにおいてはガラクトース誘導的な発現を全く示さないことが知られているが、本発明者らは、このプロモーターをクルイベロマイセス・マルシアヌスにおいて用いると、非常に意外なことに、ガラクトース誘導的な発現を示すことを見い出した。 Furthermore, the TDH3 promoter from Saccharomyces cerevisiae is known to show no galactose-inducible expression in Saccharomyces cerevisiae, but we use this promoter in Kluyveromyces marxianus. Surprisingly, it was found to show galactose-induced expression.
　本発明者らは、以上の知見に基づいて、本発明を完成するに至った。 The inventors have completed the present invention based on the above findings.
　すなわち、本発明は、（１）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子の発現方法や、（２）以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程を含むことを特徴とする上記（１）に記載の目的遺伝子の発現方法：（Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：（Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクター：や、（３）プロモーターとしてサッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用い、かつ、形質転換体の培養をガラクトース存在下で行うことを特徴とする上記（２）に記載の目的遺伝子の発現方法に関する。 That is, the present invention is characterized in that (1) a promoter selected from the group consisting of TDH3 promoter derived from Saccharomyces cerevisiae, ILV5 promoter, CWP2 promoter, and variants thereof is used in Kluyveromyces marxianus. (2) Transformation obtained by introducing the recombinant polynucleotide described in (A) below or the vector described in (B) below into Kluyveromyces marxianus A method of expressing a target gene as described in (1) above, which comprises a step of culturing the body: (A) a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof Promoters selected from the group and their control A recombinant polynucleotide comprising a target gene operably disposed below: (B) a vector comprising the recombinant polynucleotide described in (A) above; and (3) a TDH3 promoter derived from Saccharomyces cerevisiae as a promoter. And the method for expressing a target gene according to (2) above, wherein the transformant is cultured in the presence of galactose.
　また、本発明は、（４）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子産物の製造方法や、（５）以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程（Ｐ）と、培養して得られた形質転換体から目的遺伝子産物を回収する工程（Ｑ）とを含むことを特徴とする上記（４）に記載の目的遺伝子産物の製造方法：（Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：（Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクターや、（６）プロモーターとしてサッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用い、かつ、形質転換体の培養をガラクトース存在下で行うことを特徴とする上記（５）に記載の目的遺伝子産物の製造方法や、（７）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子の発現効率を上昇させる方法に関する。 In addition, the present invention is characterized in that (4) a promoter selected from the group consisting of SDH from Saccharomyces cerevisiae, a TDH3 promoter, an ILV5 promoter, a CWP2 promoter, and variants thereof is used in Kluyveromyces marxianus. And (5) a trait obtained by introducing the recombinant polynucleotide described in (A) below or the vector described in (B) below into Kluyveromyces marxianus A step (P) of culturing the transformant, and a step (Q) of recovering the target gene product from the transformant obtained by culturing. Production method: (A) TDH3 promoter, ILV5 promoter, CWP2 derived from Saccharomyces cerevisiae A recombinant polynucleotide comprising a promoter selected from the group consisting of a lomotor and a mutant thereof, and a target gene operably arranged under the control thereof: (B) the recombination according to (A) above The target gene product according to (5) above, wherein the vector comprises a polynucleotide, and (6) the TDH3 promoter derived from Saccharomyces cerevisiae is used as the promoter, and the transformant is cultured in the presence of galactose. And (7) a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, in Kluyveromyces marxianus, Increase the expression efficiency of target genes A method for.
　本発明の目的遺伝子の発現方法によると、目的遺伝子を高発現させることができる。また、本発明の目的遺伝子産物の製造方法によると、目的遺伝子産物を高効率で製造することができる。 According to the target gene expression method of the present invention, the target gene can be highly expressed. Moreover, according to the method for producing a target gene product of the present invention, the target gene product can be produced with high efficiency.
ＳｃＴＤＨ３プロモーターの発現解析試験１（グルコース培地）の結果（相対発現量（ＲＬＵ／μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / microliter)) of the expression analysis test 1 (glucose culture medium) of a ScTDH3 promoter. ＳｃＴＤＨ３プロモーターの発現解析試験１（グルコース培地）の結果（相対発現量（ＲＬＵ／ＯＤ・μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / OD * microliter)) of the expression analysis test 1 (glucose culture medium) of a ScTDH3 promoter. ＳｃＴＤＨ３プロモーターの発現解析試験２（ガラクトース培地）の結果（相対発現量（ＲＬＵ／μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / microliter)) of the expression analysis test 2 (galactose culture medium) of a ScTDH3 promoter. ＳｃＴＤＨ３プロモーターの発現解析試験２（ガラクトース培地）の結果（相対発現量（ＲＬＵ／ＯＤ・μｌ））を示す図である。It is a figure which shows the result (Relative expression level (RLU / OD * microliter)) of the expression analysis test 2 (galactose culture medium) of a ScTDH3 promoter. ＳｃＴＤＨ３プロモーターの発現解析試験３（様々な糖培地）の結果（相対発現量（ＲＬＵ／μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / microliter)) of the expression analysis test 3 (various sugar culture medium) of a ScTDH3 promoter. ＳｃＴＤＨ３プロモーターの発現解析試験３（様々な糖培地）の結果（相対発現量（ＲＬＵ／ＯＤ・μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / OD * microliter)) of the expression analysis test 3 (various sugar culture media) of a ScTDH3 promoter. サッカロマイセス・セレビシエ由来の様々なプロモーター（ＳｃＴＤＨ３プロモーター、ＳｃＩＬＶ５プロモーター、ＳｃＣＷＰ２プロモーター）の発現解析試験の結果（相対発現量（ＲＬＵ／ＯＤ・μｌ））を示す図である。It is a figure which shows the result (relative expression level (RLU / OD * microliter)) of the expression analysis test of various promoters (ScTDH3 promoter, ScILV5 promoter, ScCWP2 promoter) derived from Saccharomyces cerevisiae.
１．本発明の目的遺伝子の発現方法
　本発明の目的遺伝子の発現方法は、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーター（以下、まとめて「本発明におけるプロモーター」とも表示する。）を、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする。本発明におけるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いると、目的遺伝子を高発現させることができる。 1. Expression method of target gene of the present invention The expression method of the target gene of the present invention is a promoter selected from the group consisting of TDH3 promoter, ILV5 promoter, CWP2 promoter derived from Saccharomyces cerevisiae, and variants thereof (hereinafter summarized) Is also referred to as “promoter in the present invention”) in Kluyveromyces marxianus. When the promoter in the present invention is used in Kluyveromyces marxianus, the target gene can be highly expressed.
　本発明の目的遺伝子の発現方法のより好適な態様としては、以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程を含む。
（Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：
（Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクター： As a more preferred embodiment of the expression method of the target gene of the present invention, a recombinant polynucleotide described in (A) below or a vector described in (B) below is introduced into Kluyveromyces marxianus. Culturing the resulting transformant.
(A) A recombination comprising a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, and a target gene operably arranged under the control thereof Polynucleotide:
(B) A vector comprising the recombinant polynucleotide according to (A) above:
　上記（Ａ）における、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターとしては、配列番号１に示されるポリヌクレオチドを好適に例示することができ、サッカロマイセス・セレビシエ由来のＩＬＶ５プロモーターとしては、配列番号２に示されるポリヌクレオチドを好適に例示することができ、サッカロマイセス・セレビシエ由来のＣＷＰ２プロモーターとしては、配列番号３に示されるポリヌクレオチドを好適に例示することができる。 As the TDH3 promoter derived from Saccharomyces cerevisiae in (A) above, the polynucleotide shown in SEQ ID NO: 1 can be preferably exemplified, and as the ILV5 promoter derived from Saccharomyces cerevisiae, the polynucleotide shown in SEQ ID NO: 2 can be exemplified. Nucleotides can be preferably exemplified, and as the CWP2 promoter derived from Saccharomyces cerevisiae, the polynucleotide shown in SEQ ID NO: 3 can be suitably exemplified.
　上記（Ａ）におけるプロモーターの変異体とは、配列番号１～３のいずれかに示されるポリヌクレオチドの変異体であって、かつ、クルイベロマイセス・マルシアヌスにおいてプロモーター活性を有するポリヌクレオチドを意味する。かかるプロモーターの変異体としては、
（ａ）配列番号１～３のいずれかに示されるポリヌクレオチドに対して８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、最も好ましくは９８％以上の同一性を有し、かつ、クルイベロマイセス・マルシアヌスにおいてプロモーター活性を有するポリヌクレオチド：
（ｂ）配列番号１～３のいずれかに示されるポリヌクレオチドにおいて、１若しくは２個以上のヌクレオチドが欠失、置換若しくは付加されたポリヌクレオチドからなり、かつ、クルイベロマイセス・マルシアヌスにおいてプロモーター活性を有するポリヌクレオチド：
（ｃ）配列番号１～３のいずれかに示されるポリヌクレオチドに相補的なポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、かつ、クルイベロマイセス・マルシアヌスにおいてプロモーター活性を有するポリヌクレオチド： The variant of the promoter in the above (A) means a polynucleotide variant shown in any one of SEQ ID NOs: 1 to 3 and having a promoter activity in Kluyveromyces marxianus. . Such promoter variants include:
(A) 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more with respect to the polynucleotide shown in any one of SEQ ID NOs: 1 to 3 Polynucleotides having identity and having promoter activity in Kluyveromyces marxianus:
(B) the polynucleotide shown in any one of SEQ ID NOs: 1 to 3, consisting of a polynucleotide in which one or more nucleotides have been deleted, substituted or added, and promoter activity in Kluyveromyces marxianus A polynucleotide having:
(C) a polynucleotide that hybridizes with a polynucleotide complementary to the polynucleotide shown in any one of SEQ ID NOs: 1 to 3 under stringent conditions and has promoter activity in Kluyveromyces marxianus:
　上記（ｂ）における「１若しくは２個以上のヌクレオチドが欠失、置換若しくは付加されたポリヌクレオチド」とは、例えば１～２０個、好ましくは１～１５個、より好ましくは１～１０個、さらに好ましくは１～５個の任意の数のヌクレオチドが欠失、置換若しくは付加されたポリヌクレオチドを意味する。 The “polynucleotide in which one or two or more nucleotides are deleted, substituted or added” in the above (b) is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, Preferably, it means a polynucleotide in which any number of 1 to 5 nucleotides has been deleted, substituted or added.
　上記（ｃ）における「ストリンジェントな条件下」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいい、具体的には、８０％以上、好ましくは８５％以上の同一性を有するＤＮＡ同士がハイブリダイズし、それより同一性が低いＤＮＡ同士がハイブリダイズしない条件あるいは通常のサザンハイブリダイゼーションの洗いの条件である６５℃、１×ＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍＭ塩化ナトリウム、１５ｍＭクエン酸ナトリウム）、０．１％ＳＤＳ、又は０．１×ＳＳＣ、０．１％ＳＤＳに相当する塩濃度でハイブリダイズする条件を挙げることができる。ハイブリダイゼーションは、モレキュラークローニング第２版等に記載されている方法に準じて行うことができる。上記（ｃ）における「ストリンジェントな条件下でハイブリダイズするポリヌクレオチド」としては、プローブとして使用するポリヌクレオチドと一定以上の同一性を有するポリヌクレオチドが挙げることができ、例えば８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、最も好ましくは９８％以上の同一性を有するポリヌクレオチドを好適に例示することができる。 The “stringent condition” in the above (c) means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Specifically, it is 80% or more, preferably 85% or more. The DNAs having the same identity are hybridized with each other and DNAs with lower identity are not hybridized with each other, or a normal Southern hybridization washing condition at 65 ° C., 1 × SSC solution (1 × concentration SSC) The composition of the solution can include conditions for hybridization at a salt concentration corresponding to 150 mM sodium chloride, 15 mM sodium citrate), 0.1% SDS, or 0.1 × SSC, 0.1% SDS. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition. Examples of the “polynucleotide hybridizing under stringent conditions” in the above (c) include a polynucleotide having a certain identity or more with a polynucleotide used as a probe, for example, 80% or more, preferably Suitable examples include polynucleotides having 85% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98% or more.
　上記の配列番号１～３のいずれかに示されるポリヌクレオチドは、例えば、鋳型としてサッカロマイセス・セレビシエのゲノムＤＮＡを用い、該ヌクレオチド配列に基づいて合成したオリゴヌクレオチドをプライマーに用いるＰＣＲ反応によって、または該ヌクレオチド配列に基づいて合成したオリゴヌクレオチドをプローブとして用いるハイブリダイゼーションによっても得ることができる。なお、染色体ＤＮＡは、常法（例えば、特開２００８－２３７０２４号公報）に開示された方法により取得できる。 The polynucleotide shown in any one of the above SEQ ID NOs: 1 to 3, for example, by a PCR reaction using Saccharomyces cerevisiae genomic DNA as a template and an oligonucleotide synthesized based on the nucleotide sequence as a primer, or It can also be obtained by hybridization using an oligonucleotide synthesized based on the nucleotide sequence as a probe. Chromosomal DNA can be obtained by a method disclosed in a conventional method (for example, JP-A-2008-237024).
　オリゴヌクレオチドの合成は、例えば、市販されている種々のＤＮＡ合成機を用いて常法に従って合成できる。また、ＰＣＲ反応は、アプライドバイオシステムズ社（Applied Biosystems）製のサーマルサイクラーＧｅｎｅ ＡｍｐＰＣＲＳｙｓｔｅｍ ２４００を用い、ＴａｑＤＮＡポリメラーゼ（タカラバイオ社製）やＫＯＤ－Ｐｌｕｓ－（東洋紡績社製）などを使用して常法に従って行なうことができる。 Oligonucleotides can be synthesized according to a conventional method using various commercially available DNA synthesizers, for example. The PCR reaction is carried out using a thermal cycler Gene AmpPCRSystem 2400 manufactured by Applied Biosystems, using TaqDNA polymerase (manufactured by Takara Bio Inc.) or KOD-Plus- (manufactured by Toyobo Co., Ltd.). Can be done according to
　また、前述のポリヌクレオチドの変異体は、化学合成、遺伝子工学的手法、突然変異誘発などの当業者に既知の任意の方法により作製することもできる。具体的には、配列番号１～３のいずれかに示されるポリヌクレオチドに対し、変異原となる薬剤と接触作用させる方法、紫外線を照射する方法、遺伝子工学的な手法等を用いて、これらポリヌクレオチドに変異を導入することにより、ポリヌクレオチドの変異体を取得することができる。遺伝子工学的手法の一つである部位特異的変異誘発法は特定の位置に特定の変異を導入できる手法であることから有用であり、モレキュラークローニング第２版、Current Protocols in Molecular Biology, Supplement 1～38,John Wiley & Sons (1987-1997)等に記載の方法に準じて行うことができる。 The above-described polynucleotide variants can also be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, the polynucleotide shown in any one of SEQ ID NOs: 1 to 3 is contacted with a mutagen agent, irradiated with ultraviolet rays, genetic engineering techniques, etc. By introducing mutations into nucleotides, polynucleotide variants can be obtained. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning 2nd edition, Current Protocols in MolecularlogyBiology, Supplement 1 ～ 38, John Wiley and Sons (1987-1997).
　本発明におけるプロモーターは、クルイベロマイセス・マルシアヌスにおいてプロモーター活性を有する。あるポリヌクレオチドがクルイベロマイセス・マルシアヌスにおいてプロモーター活性を有するかどうかは、例えばそのポリヌクレオチドの下流にレポーター遺伝子を作動可能に連結した組換えポリヌクレオチドを作製する工程、その組換えポリヌクレオチドをクルイベロマイセス・マルシアヌスに形質転換して形質転換酵母を得る工程、その形質転換酵母におけるそのレポーター遺伝子の発現の程度を測定する工程を含む周知のレポーターアッセイ等により容易に確認することができる。 The promoter in the present invention has promoter activity in Kluyveromyces marxianus. Whether a polynucleotide has promoter activity in Kluyveromyces marxianus is determined by, for example, producing a recombinant polynucleotide in which a reporter gene is operably linked downstream of the polynucleotide, This can be easily confirmed by a well-known reporter assay including a step of transforming Iberomyces marcianus to obtain transformed yeast and a step of measuring the expression level of the reporter gene in the transformed yeast.
　本発明の目的遺伝子の発現方法における「高発現」や、後述の本発明の目的遺伝子産物の製造方法における「高効率」には、本発明におけるプロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結した組換えポリヌクレオチドをクルイベロマイセス・マルシアヌスに導入して得られた形質転換体を、ＹＰＤ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて２８℃、４８時間振盪培養したときの培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／ＯＤ・μｌ）が、１００００以上、好ましくは１２０００以上、より好ましくは１４０００以上、さらに好ましくは１６０００以上、より好ましくは１８０００以上であることを好適に含む。また、上記の「高発現」や、「高効率」は、上記培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／μｌ）が、３０万以上、好ましくは４５万以上、より好ましくは６０万以上、さらに好ましくは７５万以上であることも、好適に含む。 For “high expression” in the expression method of the target gene of the present invention and “high efficiency” in the method of producing the target gene product of the present invention described later, the secretory luciferase CLuc gene can be operated downstream of the promoter in the present invention. A transformant obtained by introducing a recombinant polynucleotide ligated to Kluyveromyces marxianus into a YPD liquid medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose) at 28 ° C. , The relative expression level (RLU / OD · μl) of the secreted luciferase CLuc in the culture medium when shaken for 48 hours is 10,000 or more, preferably 12,000 or more, more preferably 14,000 or more, more preferably 16000 or more, more Preferably it is preferably 18000 or more. In addition, the above-mentioned “high expression” and “high efficiency” indicate that the relative expression level (RLU / μl) of the secreted luciferase CLuc in the culture medium is 300,000 or more, preferably 450,000 or more, more preferably 60 It is suitably included that it is 10,000 or more, more preferably 750,000 or more.
　本発明の目的遺伝子の発現方法おける「高発現」や、後述の本発明の目的遺伝子産物の製造方法における「高効率」の他の例としては、本発明におけるプロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結した組換えポリヌクレオチドをクルイベロマイセス・マルシアヌスに導入して得られた形質転換体を、ＹＰＤ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて２８℃、４８時間振盪培養したときの培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／ＯＤ・μｌ）が、上記組換えポリヌクレオチドをサッカロマイセス・セレビシエに導入して得られた形質転換体を、ＹＰＤ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて２８℃、４８時間振盪培養したときの培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／ＯＤ・μｌ）に対して、割合として、３倍以上、好ましくは４倍以上、より好ましくは５倍以上、さらに好ましくは７倍以上、より好ましくは１５倍以上、さらに好ましくは２５倍以上、さらに好ましくは５０倍以上であることを好適に含む。 Other examples of “high expression” in the expression method of the target gene of the present invention and “high efficiency” in the method of producing the target gene product of the present invention described later include a secreted luciferase CLuc downstream of the promoter in the present invention. A transformant obtained by introducing a recombinant polynucleotide operably linked with a gene into Kluyveromyces marxianus was transformed into YPD liquid medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose). The relative expression level (RLU / OD · μl) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C. for 48 hours at 37 ° C. was obtained by introducing the recombinant polynucleotide into Saccharomyces cerevisiae Convert the transformant to 2 in YPD liquid medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose). The ratio is 3 times or more, preferably 4 times or more, more preferably 5 times the relative expression level (RLU / OD · μl) of the secreted luciferase CLuc in the culture medium when shaken at 48 ° C. for 48 hours. More preferably, it includes 7 times or more, more preferably 15 times or more, further preferably 25 times or more, and further preferably 50 times or more.
　本発明の目的遺伝子の発現方法おける「高発現」や、後述の本発明の目的遺伝子産物の製造方法における「高効率」のさらなる他の例としては、本発明におけるプロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結した組換えポリヌクレオチドをクルイベロマイセス・マルシアヌスに導入して得られた形質転換体を、ＹＰＧａｌ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％ガラクトース）にて２８℃、４８時間振盪培養したときの培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／ＯＤ・μｌ）が、上記組換えポリヌクレオチドをクルイベロマイセス・マルシアヌスに導入して得られた形質転換体を、ＹＰＤ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて２８℃、４８時間振盪培養したときの培養液中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／ＯＤ・μｌ）に対して、割合として、３倍以上、好ましくは４倍以上であることを好適に含む。 As yet another example of “high expression” in the method for expressing the target gene of the present invention and “high efficiency” in the method for producing the target gene product of the present invention described later, a secretory luciferase is provided downstream of the promoter in the present invention. A transformant obtained by introducing a recombinant polynucleotide operably linked to a CLuc gene into Kluyveromyces marxianus was transformed into a YPGal liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass galactose. ), The relative expression level (RLU / OD · μl) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C. for 48 hours introduced the recombinant polynucleotide into Kluyveromyces marxianus. The obtained transformant was treated with YPD liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass). The ratio is 3 times or more, preferably 4 times or more relative to the relative expression level (RLU / OD · μl) of the secreted luciferase CLuc in the culture medium after shaking culture at 28 ° C. for 48 hours at It is preferably included.
　本発明におけるポリヌクレオチドとしては、相補的な二本鎖ポリヌクレオチドを好適に例示することができ、中でも相補的な二本鎖ＤＮＡを特に好適に例示することができる。 As the polynucleotide in the present invention, a complementary double-stranded polynucleotide can be preferably exemplified, and a complementary double-stranded DNA can be particularly preferably exemplified.
　上記（Ａ）の組換えポリヌクレオチドの「本発明におけるプロモーターの制御下に作動可能に配置された目的遺伝子」とは、本発明のプロモーターに転写因子が結合することにより、その目的遺伝子の発現が誘導されるように、本発明のプロモーターと、その目的遺伝子とが連結されていることを意味する。上記「目的遺伝子」としては、任意の遺伝子であればよいが、なんらかの有用なタンパク質をコードする有用タンパク質遺伝子を好適に例示することができる。有用タンパク質遺伝子としては、セルラーゼ遺伝子、アミラーゼ遺伝子等の糖化酵素遺伝子や、ウイルスワクチンタンパク質の遺伝子を好適に例示することができる。 The “target gene operably arranged under the control of the promoter in the present invention” of the recombinant polynucleotide (A) above means that the expression of the target gene is caused by binding of a transcription factor to the promoter of the present invention. It means that the promoter of the present invention and its target gene are linked so as to be induced. The “target gene” may be any gene, and a useful protein gene encoding any useful protein can be preferably exemplified. Preferable examples of useful protein genes include saccharifying enzyme genes such as cellulase gene and amylase gene, and viral vaccine protein genes.
　上記（Ｂ）記載の本発明におけるベクターは、上記の本発明における組換えポリヌクレオチドを含む。本発明におけるベクターを用いると、クルイベロマイセス・マルシアヌスにおいて、本発明における組換えポリヌクレオチドを保持したり、目的遺伝子を発現させるためにクルイベロマイセス・マルシアヌスを形質転換したりすることができる。本発明におけるベクターは、直鎖状であってもよいし、環状であってもよい。クルイベロマイセス・マルシアヌスは、直鎖状のベクターであっても染色体上において高頻度で組換えを生じさせることができ、その結果、形質転換することができる。また、環状のベクターの場合、さらに自己複製配列を含んでいれば、該ベクターはクルイベロマイセス・マルシアヌス細胞内にて自律複製し、酵母細胞内で保持され、その結果、形質転換することができる。 The vector according to the present invention described in (B) above includes the recombinant polynucleotide according to the present invention. When the vector of the present invention is used, Kluyveromyces marcianus can be used to retain the recombinant polynucleotide of the present invention or to transform kluyveromyces marcianus to express the target gene. . The vector in the present invention may be linear or circular. Kluyveromyces marxianus can cause recombination at high frequency on a chromosome even if it is a linear vector, and as a result, can be transformed. In the case of a circular vector, if the vector further contains a self-replicating sequence, the vector autonomously replicates in the Kluyveromyces marxianus cell and is retained in the yeast cell. As a result, the vector can be transformed. it can.
　上記ベクターは、クルイベロマイセス・マルシアヌス細胞内で目的遺伝子を発現させることが可能であるものである限り特に制限されず、環状のプラスミドベクターとしては、例えばｐＫＤ１等を好適に例示することができる。 The vector is not particularly limited as long as the target gene can be expressed in Kluyveromyces marxianus cells. As a circular plasmid vector, for example, pKD1 can be preferably exemplified. .
　本発明における形質転換体は、本発明における組換えポリヌクレオチド、又は、本発明におけるベクターを、クルイベロマイセス・マルシアヌスに導入することにより得られる。本発明における形質転換体は、その細胞内で目的遺伝子を高発現することができる。 The transformant in the present invention can be obtained by introducing the recombinant polynucleotide in the present invention or the vector in the present invention into Kluyveromyces marxianus. The transformant in the present invention can highly express the target gene in the cell.
　本発明における組換えポリヌクレオチド、又は、本発明におけるベクターの酵母への導入方法としては、特に制限されず、ウイルスベクターを利用する方法、特異的受容体を利用する方法、細胞融合法等の生物学的方法；エレクトロポレーション法、マイクロインジェクション法、遺伝子銃法、超音波遺伝子導入法等の物理的方法；リポフェクション法、リン酸カルシウム共沈殿法、リポソーム法、ＤＥＡＥデキストラン法等の化学的方法；などの公知の方法を例示することができ、中でも簡便であって汎用性が高い点でリポフェクション法を好適に例示することができる。また、本発明における組換えポリヌクレオチド、又は、本発明におけるベクターがその酵母へ導入されたかどうかは、目的遺伝子として、又は、目的遺伝子に加えて、マーカー遺伝子を本発明における組換えポリヌクレオチドや本発明におけるベクターにマーカー遺伝子を挿入しておき、形質転換体におけるそのマーカー遺伝子の発現を確認するなどして容易に確認することができる。 The method for introducing the recombinant polynucleotide in the present invention or the vector in the present invention into yeast is not particularly limited, and organisms such as a method using a viral vector, a method using a specific receptor, a cell fusion method, etc. Physical methods such as electroporation method, microinjection method, gene gun method, ultrasonic gene transfer method; chemical methods such as lipofection method, calcium phosphate coprecipitation method, liposome method, DEAE dextran method, etc. Known methods can be exemplified, and among them, the lipofection method can be suitably exemplified in terms of simplicity and high versatility. In addition, whether the recombinant polynucleotide in the present invention or the vector in the present invention has been introduced into the yeast depends on the marker gene as the target gene or in addition to the target gene. It can be easily confirmed by inserting a marker gene into the vector of the invention and confirming the expression of the marker gene in the transformant.
　本発明の目的遺伝子の発現方法のより好適な態様としては、前述したように、本発明における形質転換体を培養する工程を含むことを特徴とする。「本発明における形質転換体を培養する方法」としては、その形質転換体が増殖し得る限り特に制限されないが、例えばその形質転換体が増殖可能な温度条件下（例えば２５～５０℃、好ましくは２５～４０℃、より好ましくは２８～３３℃）、ＹＰＤ培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて、適当な時間（例えば１～１０日間、好ましくは１～５日間、より好ましくは１～３日間）、振盪培養する方法を好適に例示することができる。なお、本発明におけるプロモーターのうち、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用いた場合は、目的遺伝子をより顕著に高発現させ得ることから、糖源として、グルコースに代えて、ガラクトースを用いることが好ましい。すなわち、本発明におけるプロモーターのうち、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用いた場合は、得られた形質転換体の培養を、ガラクトース存在下で行うことが好ましい。 As a more preferred embodiment of the target gene expression method of the present invention, as described above, the method includes the step of culturing the transformant of the present invention. The “method for culturing the transformant in the present invention” is not particularly limited as long as the transformant can grow. For example, the transformant can be grown under temperature conditions (for example, 25 to 50 ° C., preferably 25 to 40 ° C., more preferably 28 to 33 ° C.) and YPD medium (1% by weight yeast extract, 2% by weight polypeptone, 2% by weight glucose) for an appropriate time (eg 1 to 10 days, preferably 1 to A method of shaking culture for 5 days, more preferably 1 to 3 days) can be suitably exemplified. Of the promoters in the present invention, when the TDH3 promoter derived from Saccharomyces cerevisiae is used, the target gene can be expressed more significantly, and therefore it is preferable to use galactose instead of glucose as the sugar source. . That is, among the promoters in the present invention, when the TDH3 promoter derived from Saccharomyces cerevisiae is used, the obtained transformant is preferably cultured in the presence of galactose.
２．本発明の目的遺伝子産物の製造方法
　本発明の目的遺伝子産物の製造方法は、本発明におけるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする。本発明におけるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いると、目的遺伝子産物を高効率で製造することができる。 2. Method for producing target gene product of the present invention The method for producing a target gene product of the present invention is characterized in that the promoter of the present invention is used in Kluyveromyces marxianus. When the promoter of the present invention is used in Kluyveromyces marcianus, the target gene product can be produced with high efficiency.
　本発明の目的遺伝子産物の製造方法のより好適な態様としては、以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程（Ｐ）と、培養して得られた形質転換体から目的遺伝子産物を回収する工程（Ｑ）とを含む。
（Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：
（Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクター： As a more preferred embodiment of the method for producing a target gene product of the present invention, a recombinant polynucleotide described in (A) below or a vector described in (B) below is introduced into Kluyveromyces marxianus. A step (P) of culturing the transformant obtained in this manner, and a step (Q) of recovering the target gene product from the transformant obtained by culturing.
(A) A recombination comprising a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, and a target gene operably arranged under the control thereof Polynucleotide:
(B) A vector comprising the recombinant polynucleotide according to (A) above:
　本発明の目的遺伝子産物の製造方法のより好適な態様における工程（Ｐ）は、前述の本発明の目的遺伝子の発現方法のより好適な態様における工程と同じである。また、上記工程（Ｑ）における「培養して得られた形質転換体から目的遺伝子産物を回収する」方法としては、目的遺伝子産物を回収し得る限り特に制限されず、クロマトグラフィーを利用した方法、タグを利用した方法などの公知の方法を例示することができる。 The step (P) in the more preferable embodiment of the method for producing the target gene product of the present invention is the same as the step in the more preferable embodiment of the aforementioned target gene expression method of the present invention. The method of “recovering the target gene product from the transformant obtained by culturing” in the above step (Q) is not particularly limited as long as the target gene product can be recovered, and a method using chromatography, A known method such as a method using a tag can be exemplified.
３．本発明の目的遺伝子の発現効率を上昇させる方法
　本発明の目的遺伝子の発現効率を上昇させる方法は、本発明におけるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする。本発明におけるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いると、目的遺伝子の発現効率を向上させることができる。本発明の目的遺伝子の発現効率を上昇させる方法における好適な態様としては、前述の本発明の目的遺伝子の発現方法における好適な態様と同様の態様を例示することができる。 3. Method for increasing the expression efficiency of the target gene of the present invention The method for increasing the expression efficiency of the target gene of the present invention is characterized in that the promoter of the present invention is used in Kluyveromyces marxianus. When the promoter of the present invention is used in Kluyveromyces marxianus, the expression efficiency of the target gene can be improved. As a preferable aspect in the method for increasing the expression efficiency of the target gene of the present invention, the same aspect as the preferable aspect in the aforementioned expression method of the target gene of the present invention can be exemplified.
［サッカロマイセス・セレビシエからのプロモーターの単離］
　公知の配列情報に基づき、サッカロマイセス・セレビシエからのＴＤＨ３プロモーター（ＳｃＴＤＨ３プロモーター）、ＩＬＶ５プロモーター（ＳｃＩＬＶ５プロモーター）、ＣＷＰ２プロモーター（ＳｃＣＷＰ２プロモーター）をそれぞれ単離した。ＳｃＴＤＨ３プロモーターの配列は配列番号１に示され、ＳｃＩＬＶ５プロモーターの配列は配列番号２に示され、ＳｃＣＷＰ２プロモーターの配列は配列番号３に示される。 [Isolation of promoter from Saccharomyces cerevisiae]
Based on the known sequence information, TDH3 promoter (ScTDH3 promoter), ILV5 promoter (ScILV5 promoter), and CWP2 promoter (ScCWP2 promoter) from Saccharomyces cerevisiae were isolated. The sequence of the ScTDH3 promoter is shown in SEQ ID NO: 1, the sequence of the ScILV5 promoter is shown in SEQ ID NO: 2, and the sequence of the ScCWP2 promoter is shown in SEQ ID NO: 3.
［ＳｃＴＤＨ３プロモーターの発現解析試験１（グルコース培地）］
　サッカロマイセス・セレビシエ由来のＳｃＴＤＨ３プロモーターが、クルイベロマイセス・マルシアヌスにおいて目的遺伝子を発現させることができるかどうかを調べるために、以下のような発現解析試験を行った。 [ScTDH3 promoter expression analysis test 1 (glucose medium)]
In order to examine whether the ScTDH3 promoter derived from Saccharomyces cerevisiae can express the target gene in Kluyveromyces marxianus, the following expression analysis test was performed.
　まずは以下の２つの形質転換体を、公知の形質転換法により、作製した。
（１）ＳｃＴＤＨ３プロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結したＳｃＴＤＨ３ｐ－ＣＬｕｃをサッカロマイセス・セレビシエに導入して得られた比較例形質転換体１（Ｓｃ　ＴＦ　ＳｃＴＤＨ３ｐ－ＣＬｕｃ）。
（２）上記のＳｃＴＤＨ３ｐ－ＣＬｕｃをクルイベロマイセス・マルシアヌスに導入して得られた実施例形質転換体１（Ｋｍ　ＴＦ　ＳｃＴＤＨ３ｐ－ＣＬｕｃ）。 First, the following two transformants were prepared by a known transformation method.
(1) A comparative transformant 1 (Sc TF ScTDH3p-CLuc) obtained by introducing ScTDH3p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScTDH3 promoter into Saccharomyces cerevisiae.
(2) Example transformant 1 (Km TF ScTDH3p-CLuc) obtained by introducing the above ScTDH3p-CLuc into Kluyveromyces marxianus.
　これらの両形質転換体を、それぞれＹＰＤ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％グルコース）にて２８℃で振盪培養した。その培養液を経時的にサンプリングして、培養液１μｌ中の分泌型ルシフェラーゼＣＬｕｃの相対発現量（ＲＬＵ／μｌ）、及び、培養液のＯＤ６００を測定した。培養開始直後、培養開始から２４時間経過後、４８時間経過後、７２時間経過後、９６時間経過後、１２０時間経過後、及び、１４４時間経過後における測定結果（相対発現量（ＲＬＵ／μｌ））を図１に示す。なお、図１のグラフは、各種の形質転換体につき、３クローンずつの平均値で示した。また、上記相対発現量（ＲＬＵ／μｌ）の値を、培地のＯＤ６００の測定値で割って算出した値（相対発現量（ＲＬＵ／ＯＤ・μｌ））で表した結果を図２に示す。図１及び図２の結果から分かるように、実施例形質転換体１は、比較例形質転換体１に比べて、早期かつ顕著な高発現性を示した。特に、実施例形質転換体１は、比較例形質転換体１と比較して、培養開始直後から高発現性を示し、培養開始から４８時間経過後には、実施例形質転換体１は、比較例形質転換体１に比べて、割合として、１３９倍以上もの相対発現量（ＲＬＵ／μｌ）（６６倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示した。実施例形質転換体１のこの「早期かつ顕著な高発現性」は、目的遺伝子産物等の単位時間当たりの製造効率を飛躍的に高めることとなるので、顕著な効果といえる。 Both of these transformants were each cultured with shaking at 28 ° C. in a YPD liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass glucose). The culture solution was sampled over time, and the relative expression level (RLU / μl) of the secreted luciferase CLuc in 1 μl of the culture solution and the OD600 of the culture solution were measured. Measurement results (relative expression level (RLU / μl)) immediately after the start of culture, 24 hours after the start of culture, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours ) Is shown in FIG. The graph of FIG. 1 shows the average value of 3 clones for each type of transformant. FIG. 2 shows the result of expressing the value of the relative expression level (RLU / μl) divided by the measured value of OD600 of the medium (relative expression level (RLU / OD · μl)). As can be seen from the results of FIGS. 1 and 2, Example Transformant 1 showed early and remarkable high expression compared to Comparative Example Transformant 1. In particular, the Example transformant 1 showed high expression immediately after the start of the culture as compared with the Comparative Example transformant 1, and after 48 hours from the start of the culture, the Example transformant 1 Compared to transformant 1, the relative expression level (RLU / μl) of 139 times or more (relative expression level (RLU / OD · μl) of 66 times or more) was shown as a ratio. This “early and remarkable high expression” of Example transformant 1 can be said to be a remarkable effect because it greatly increases the production efficiency per unit time of the target gene product and the like.
　以上のように、サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター（ＳｃＴＤＨ３プロモーター）は、サッカロマイセス・セレビシエにおいて用いた場合に比べて、クルイベロマイセス・マルシアヌスにおいて用いた場合に、早期かつ顕著な高発現性を示した。一般的に、プロモーターは、そのプロモーターが由来する生物種の細胞内において最も高いプロモーター活性を示し、他の生物種においては、プロモーター活性そのものを示さないか、あるいは、低下したプロモーター活性を示すことを考慮すると、本発現解析試験の結果は意外であった。なお、本明細書中の「活性量」は「相対発現量」と同義である。 As described above, the TDH3 promoter derived from Saccharomyces cerevisiae (ScTDH3 promoter) exhibits early and remarkable high expression when used in Kluyveromyces marcianus compared to that used in Saccharomyces cerevisiae. It was. In general, a promoter exhibits the highest promoter activity in the cell of the species from which the promoter is derived, and in other species it does not exhibit promoter activity itself or exhibits reduced promoter activity. Considering this, the results of this expression analysis test were unexpected. In the present specification, “activity amount” is synonymous with “relative expression level”.
［ＳｃＴＤＨ３プロモーターの発現解析試験２（ガラクトース培地）］
　実施例形質転換体１の早期かつ顕著な高発現性が、他の種類の糖を用いた場合にも得られるかどうか確認するために、上記実施例２における発現解析試験において、ＹＰＤ液体培地に代えて、ＹＰＧａｌ液体培地（１質量％酵母エキス、２質量％ポリペプトン、２質量％ガラクトース）を用いたこと以外は同じ方法で、発現解析を行った。その結果を図３に示す。また、その発現解析で得られた相対発現量（ＲＬＵ／μｌ）の値を、培地のＯＤ６００の測定値で割って算出した値（相対発現量（ＲＬＵ／ＯＤ・μｌ））で表した結果を図４に示す。図３及び図４の結果から分かるように、グルコース培地に代えてガラクトース培地を用いると、実施例形質転換体１は、より顕著な高発現性を示した。例えば、実施例形質転換体１は、ガラクトース培地条件下では、グルコース培地条件下に比べて、培養開始から４８時間経過後には４．９倍以上もの相対発現量（ＲＬＵ／μｌ）（５．１倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示し、１２０時間経過後には４．９倍以上もの相対発現量（ＲＬＵ／μｌ）（４．８倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示した。ＳｃＴＤＨ３プロモーターは、サッカロマイセス・セレビシエにおいてガラクトース誘導性を全く示さないことからすると、本発現解析試験の結果はきわめて意外であった。
　なお、ガラクトース培地条件下における実施例形質転換体１は、比較例形質転換体１に比べて、培養開始から４８時間経過後には８３３倍以上もの相対発現量（ＲＬＵ／μｌ）（６０７倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示し、１２０時間経過後には８６倍以上もの相対発現量（ＲＬＵ／μｌ）（７２倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示した。 [ScTDH3 promoter expression analysis test 2 (galactose medium)]
In order to confirm whether early and remarkable high expression of Example transformant 1 can be obtained even when other types of sugars are used, in the expression analysis test in Example 2 above, in the YPD liquid medium, Instead, expression analysis was performed in the same manner except that YPGaal liquid medium (1% by mass yeast extract, 2% by mass polypeptone, 2% by mass galactose) was used. The result is shown in FIG. In addition, the value expressed by the relative expression level (RLU / μl) obtained by the expression analysis divided by the measured value of OD600 of the medium (relative expression level (RLU / OD · μl)) As shown in FIG. As can be seen from the results of FIG. 3 and FIG. 4, when a galactose medium was used instead of the glucose medium, the Example transformant 1 showed a more remarkable high expression. For example, in the transformant of Example 1, the relative expression level (RLU / μl) (5.1 times or more) under the condition of galactose medium is 4.9 times or more after 48 hours from the start of the culture as compared with the condition of glucose medium. Fold relative expression level (RLU / OD · μl)), and after 120 hours, 4.9-fold relative expression level (RLU / μl) (4.8-fold relative expression level (RLU / OD)) -Μl)). The results of this expression analysis test were very surprising given that the ScTDH3 promoter does not show any galactose inducibility in Saccharomyces cerevisiae.
In addition, the Example transformant 1 under the galactose medium condition was 833 times or more relative expression level (RLU / μl) (607 times or more) after 48 hours from the start of the culture, compared with the comparative example transformant 1. Relative expression level (RLU / OD · μl)) was shown, and after 120 hours, 86 times or more relative expression level (RLU / μl) (72 times or more relative expression level (RLU / OD · μl)) was shown. .
［ＳｃＴＤＨ３プロモーターの発現解析試験３（様々な糖培地）］
　上記の実施例形質転換体１が、様々な糖培地を用いた場合にどのような発現性を示すかどうかを調べるために、実施例形質転換体１を用いた発現解析試験を行った。その発現解析試験の方法としては、上記実施例２における発現解析試験において、ＹＰＤ液体培地中のグルコースに代えて、ガラクトース、フルクトース、又は、マンノースを同質量用いたこと以外は同じ方法を用いた。なお、ＹＰＤ液体培地中のグルコースに代えてガラクトースを同質量用いた培地はＹＰＧａｌ液体培地となる。今回の発現解析において、培養開始から４８時間経過後に測定した相対発現量（ＲＬＵ／μｌ）の結果を図５に示す。また、その発現解析で得られた相対発現量（ＲＬＵ／μｌ）の値を、培地のＯＤ６００の測定値で割って算出した値（相対発現量（ＲＬＵ／ＯＤ・μｌ））で表した結果を図６に示す。 [ScTDH3 promoter expression analysis test 3 (various sugar media)]
An expression analysis test using the Example transformant 1 was performed in order to examine how the above-described Example transformant 1 exhibited expression when various sugar media were used. As the method for the expression analysis test, the same method was used except that the same amount of galactose, fructose, or mannose was used in place of glucose in the YPD liquid medium in the expression analysis test in Example 2 above. A medium using the same mass of galactose instead of glucose in the YPD liquid medium is a YPGal liquid medium. In this expression analysis, the results of relative expression levels (RLU / μl) measured 48 hours after the start of culture are shown in FIG. In addition, the value expressed by the relative expression level (RLU / μl) obtained by the expression analysis divided by the measured value of OD600 of the medium (relative expression level (RLU / OD · μl)) As shown in FIG.
　図５及び図６の結果から分かるように、フルクトースやマンノースを用いた場合は、グルコースを用いた場合と同程度の相対発現量を示した。ガラクトースを用いた場合にのみ、他の糖を用いた場合と比較して、約５倍以上もの相対発現量（ＲＬＵ／μｌ）（約４．５倍以上もの相対発現量（ＲＬＵ／ＯＤ・μｌ））を示した。この結果から、ＳｃＴＤＨ３プロモーターをクルイベロマイセス・マルシアヌスにおいて用いたときのガラクトース誘導性は、ガラクトースに特異的な誘導性であることが示された。 As can be seen from the results of FIGS. 5 and 6, when fructose or mannose was used, the relative expression level was the same as when glucose was used. Only when galactose is used, a relative expression level (RLU / μl) of about 5 times or more (relative expression level (RLU / OD · μl) of about 4.5 times or more) compared to the case of using other sugars. ))showed that. From this result, it was shown that the galactose inducibility when the ScTDH3 promoter was used in Kluyveromyces marxianus was specific to galactose.
［サッカロマイセス・セレビシエ由来の様々なプロモーターの発現解析試験］
　ＳｃＴＤＨ３プロモーター以外の、サッカロマイセス・セレビシエ由来のプロモーターでも同様の効果が得られるかどうかを調べるために、以下のような発現解析試験を行った。 [Expression analysis test of various promoters derived from Saccharomyces cerevisiae]
In order to investigate whether the same effect can be obtained with promoters derived from Saccharomyces cerevisiae other than the ScTDH3 promoter, the following expression analysis test was conducted.
　以下の４つの形質転換体を、公知の形質転換法により、作製した。
（３）ＳｃＩＬＶ５プロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結したＳｃＩＬＶ５ｐ－ＣＬｕｃをサッカロマイセス・セレビシエに導入して得られた比較例形質転換体２（Ｓｃ　ＴＦ　ＳｃＩＬＶ５ｐ－ＣＬｕｃ）。
（４）上記のＳｃＩＬＶ５ｐ－ＣＬｕｃをクルイベロマイセス・マルシアヌスに導入して得られた実施例形質転換体２（Ｋｍ　ＴＦ　ＳｃＩＬＶ５ｐ－ＣＬｕｃ）。
（５）ＳｃＣＷＰ２プロモーターの下流に、分泌型ルシフェラーゼＣＬｕｃ遺伝子を作動可能に連結したＳｃＣＷＰ２ｐ－ＣＬｕｃをサッカロマイセス・セレビシエに導入して得られた比較例形質転換体３（Ｓｃ　ＴＦ　ＳｃＣＷＰ２ｐ－ＣＬｕｃ）。
（６）上記のＳｃＣＷＰ２ｐ－ＣＬｕｃをクルイベロマイセス・マルシアヌスに導入して得られた実施例形質転換体３（Ｋｍ　ＴＦ　ＳｃＣＷＰ２ｐ－ＣＬｕｃ）。 The following four transformants were produced by a known transformation method.
(3) Comparative transformant 2 (Sc TF ScILV5p-CLuc) obtained by introducing ScILV5p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScILV5 promoter into Saccharomyces cerevisiae.
(4) Example transformant 2 (Km TF ScILV5p-CLuc) obtained by introducing the above ScILV5p-CLuc into Kluyveromyces marcianus.
(5) Comparative transformant 3 (Sc TF ScCWP2p-CLuc) obtained by introducing ScCWP2p-CLuc operably linked with a secreted luciferase CLuc gene downstream of the ScCWP2 promoter into Saccharomyces cerevisiae.
(6) Example transformant 3 (Km TF ScCWP2p-CLuc) obtained by introducing the above ScCWP2p-CLuc into Kluyveromyces marxianus.
　ＳｃＴＤＨ３プロモーターを用いた比較例形質転換体１及び実施例形質転換体１に加えて、上記の比較例形質転換体２～３及び実施例形質転換体２～３を用いて、上記実施例２における発現解析試験と同様の発現解析試験を行った。培養開始から４８時間経過後に測定した相対発現量（ＲＬＵ／ＯＤ・μｌ）の結果を図７に示す。なお、図７には、左から順に、比較例形質転換体１、実施例形質転換体１、比較例形質転換体２、実施例形質転換体２、比較例形質転換体３、実施例形質転換体３の結果を示す。 In addition to the comparative example transformant 1 and the example transformant 1 using the ScTDH3 promoter, the above comparative example transformant 2 to 3 and the example transformant 2 to 3 were used. An expression analysis test similar to the expression analysis test was performed. The result of the relative expression level (RLU / OD · μl) measured 48 hours after the start of culture is shown in FIG. In FIG. 7, in order from the left, Comparative Example Transformant 1, Example Transformant 1, Comparative Example Transformant 2, Example Transformant 2, Comparative Example Transformant 3, and Example Transform The result of body 3 is shown.
　図７の結果から分かるように、実施例形質転換体１は、比較例形質転換体１に比べて１８倍以上の相対発現量（ＲＬＵ／ＯＤ・μｌ）を示し、実施例形質転換体２は、比較例形質転換体２に比べて７倍以上の相対発現量（ＲＬＵ／ＯＤ・μｌ）を示し、実施例形質転換体３は、比較例形質転換体３に比べて４倍以上の相対発現量（ＲＬＵ／ＯＤ・μｌ）を示した。また、実施例形質転換体２の相対発現量（４０８８２ＲＬＵ／ＯＤ・μｌ）、及び、実施例形質転換体３の相対発現量（２９３２４ＲＬＵ／ＯＤ・μｌ）は、いずれも、実施例形質転換体１の示した相対発現量（２８２５１ＲＬＵ／ＯＤ・μｌ）より高かった。 As can be seen from the results of FIG. 7, Example transformant 1 showed a relative expression level (RLU / OD · μl) 18 times or more that of Comparative Example transformant 1, and Example Transformant 2 was The relative expression level (RLU / OD · μl) is 7 times or more that of Comparative Example Transformant 2, and Example Transformant 3 is 4 times or more relative expression compared to Comparative Example Transformant 3. The amount (RLU / OD · μl) was indicated. In addition, the relative expression level of Example transformant 2 (40882 RLU / OD · μl) and the relative expression level of Example transformant 3 (29324 RLU / OD · μl) are both shown in Example transformant 1. Relative expression level (28251 RLU / OD · μl).
　以上のように、サッカロマイセス・セレビシエ由来のＩＬＶ５プロモーター（ＳｃＩＬＶ５プロモーター）、ＣＷＰ２プロモーター（ＳｃＣＷＰ２プロモーター）も、サッカロマイセス・セレビシエにおいて用いた場合に比べて、クルイベロマイセス・マルシアヌスにおいて用いた場合に、顕著な高発現性を示した。 As described above, the ILV5 promoter derived from Saccharomyces cerevisiae (ScILV5 promoter) and the CWP2 promoter (ScCWP2 promoter) are also more prominent when used in Kluyveromyces marxianus than in Saccharomyces cerevisiae. High expression was shown.
　本発明は、目的遺伝子の高発現や、目的遺伝子産物の高効率での製造の分野において特に有用に利用することができる。 The present invention can be used particularly effectively in the fields of high expression of a target gene and production of a target gene product with high efficiency.

Claims (7)

  1. サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子の発現方法。 A method for expressing a target gene, wherein a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and mutants thereof is used in Kluyveromyces marxianus.
  2. 以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程を含むことを特徴とする請求項１に記載の目的遺伝子の発現方法：
    （Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：
    （Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクター。     The method comprises culturing a transformant obtained by introducing the recombinant polynucleotide described in (A) below or the vector described in (B) below into Kluyveromyces marxianus. The expression method of the target gene according to claim 1:
    (A) A recombination comprising a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and variants thereof, and a target gene operably arranged under the control thereof Polynucleotide:
    (B) A vector comprising the recombinant polynucleotide described in (A) above.
  3. プロモーターとしてサッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用い、かつ、形質転換体の培養をガラクトース存在下で行うことを特徴とする請求項２に記載の目的遺伝子の発現方法。 The method for expressing a target gene according to claim 2, wherein a TDH3 promoter derived from Saccharomyces cerevisiae is used as the promoter, and the transformant is cultured in the presence of galactose.
  4. サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子産物の製造方法。 A method for producing a target gene product, wherein a promoter selected from the group consisting of a TDH3 promoter derived from Saccharomyces cerevisiae, an ILV5 promoter, a CWP2 promoter, and mutants thereof is used in Kluyveromyces marxianus.
  5. 以下の（Ａ）記載の組換えポリヌクレオチド、又は、以下の（Ｂ）記載のベクターを、クルイベロマイセス・マルシアヌスに導入して得られる形質転換体を培養する工程（Ｐ）と、培養して得られた形質転換体から目的遺伝子産物を回収する工程（Ｑ）とを含むことを特徴とする請求項４に記載の目的遺伝子産物の製造方法：
    （Ａ）サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターと、その制御下に作動可能に配置された目的遺伝子とを含む組換えポリヌクレオチド：
    （Ｂ）上記（Ａ）に記載の組換えポリヌクレオチドを含むベクター。     A step (P) of culturing a transformant obtained by introducing a recombinant polynucleotide described in (A) below or a vector described in (B) below into Kluyveromyces marcianus; And a step (Q) of recovering the target gene product from the transformant obtained as described above.
    (A) Recombination comprising a promoter selected from the group consisting of TDH3 promoter derived from Saccharomyces cerevisiae, ILV5 promoter, CWP2 promoter, and mutants thereof, and a target gene operably arranged under the control thereof Polynucleotide:
    (B) A vector comprising the recombinant polynucleotide described in (A) above.
  6. プロモーターとしてサッカロマイセス・セレビシエ由来のＴＤＨ３プロモーターを用い、かつ、形質転換体の培養をガラクトース存在下で行うことを特徴とする請求項５に記載の目的遺伝子産物の製造方法。 6. The method for producing a target gene product according to claim 5, wherein a TDH3 promoter derived from Saccharomyces cerevisiae is used as the promoter, and the transformant is cultured in the presence of galactose.
  7. サッカロマイセス・セレビシエ由来のＴＤＨ３プロモーター、ＩＬＶ５プロモーター、ＣＷＰ２プロモーター、及び、それらの変異体からなる群から選択されるプロモーターを、クルイベロマイセス・マルシアヌスにおいて用いることを特徴とする目的遺伝子の発現効率を上昇させる方法。 Increasing the expression efficiency of a target gene characterized in that a promoter selected from the group consisting of TDH3 promoter, ILV5 promoter, CWP2 promoter derived from Saccharomyces cerevisiae and variants thereof is used in Kluyveromyces marxianus How to make.
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