WO2016021149A1 - High-expression promoter - Google Patents
High-expression promoter Download PDFInfo
- Publication number
- WO2016021149A1 WO2016021149A1 PCT/JP2015/003793 JP2015003793W WO2016021149A1 WO 2016021149 A1 WO2016021149 A1 WO 2016021149A1 JP 2015003793 W JP2015003793 W JP 2015003793W WO 2016021149 A1 WO2016021149 A1 WO 2016021149A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- promoter
- yeast
- ycluc
- seq
- Prior art date
Links
Images
Classifications
-
- 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
-
- 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 relates to a promoter for high expression of a target gene in yeast, and more particularly, a promoter for high expression of a target gene in yeast, comprising a promoter sequence of yeast TDH3 or GAL10 gene, yeast
- yeast The present invention relates to a promoter which is sequentially provided with an intron sequence of a somal protein gene.
- Yeast is used as a production host for enzymes and pharmaceutical proteins, and the use of a high expression promoter is indispensable to increase the productivity of these proteins.
- various high expression promoters have been used to highly express proteins in yeast. Examples of such a promoter include a constant high expression TDH3 gene promoter (hereinafter also referred to as “TDH3 promoter”), a PGK1 gene promoter, an ADH1 gene promoter, an inducible high expression GAL1 gene promoter, and a GAL10 gene promoter ( (Hereinafter also referred to as “GAL10 promoter”), CUP1 gene promoter, PHO5 gene promoter, etc. Among them, those that can be expected to have a strong expression level are TDH3 gene promoter, PGK gene promoter, etc. Glycogen promoters are preferred.
- a promoter that is located upstream of the PIR1 gene in the chromosome of Kluyveromyces marxianus and controls the expression of the PIR1 gene is provided.
- a method of using (see Patent Document 1), or a method of using a DNA fragment comprising a non-translated region 5 ′ upstream of the 5 ′ upstream 150 base pairs of the start codon of the TDH3 gene and a foreign heterologous gene (patent) 2), a gene expression method characterized by introducing a target gene into the genome under the control of a yeast pyruvate decarboxylase gene (PDC) promoter (see Patent Document 3), an artificial intron-like A method (see Non-Patent Document 1) for highly expressing the sequence has been proposed.
- PDC yeast pyruvate decarboxylase gene
- JP 2013-179899 A Japanese Patent Laid-Open No. 08-168380 JP 2003-164295 A
- An object of the present invention is to provide a promoter with higher expression than the TDH3 promoter and the GAL10 promoter.
- the present inventors came to pay attention to the fact that introns have an effect of enhancing expression ability from genome-wide analysis using yeast gene disruption strains.
- an intron was present upstream of the start codon, it was considered that overexpression was possible without manipulating the structural gene. Therefore, a promoter sequence containing an intron upstream from the start codon or near the start codon region was searched from the genome.
- the promoter sequence of a ribosomal protein gene which is a gene encoding several ribosomal proteins (hereinafter also referred to as “RPS”), has high activity.
- the expression ability was further increased by replacing the enhancer regions of these promoter sequences with the enhancer regions of the general high expression promoter TDH3 promoter and the inducible high expression promoter GAL10 promoter. Therefore, when the TDH3 promoter sequence was placed at various positions upstream of the intron sequence of the promoter of the ribosomal protein gene using the secreted luciferase gene as a reporter gene, the expression was examined. The TDH3 promoter sequence was immediately upstream of the intron sequence. It was revealed that the luciferase activity was the highest when the was placed.
- a galactose-inducible high-expression promoter GAL10 promoter sequence is located upstream of the intron sequence of a ribosomal protein gene promoter and has a high galactose-inducible luciferase activity.
- a promoter for highly expressing a target gene in yeast comprising a yeast TDH3 or GAL10 promoter sequence and an intron sequence of a yeast ribosomal protein gene in sequence, and the yeast ribosomal A promoter, wherein the intron sequence of the protein gene is any one of the following sequences (a) to (c): (A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4; (B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4; (C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4; (2) The promoter according to (1) above, wherein the yeast is Saccharomyces cerevisiae. (3) A recombinant vector comprising the promoter according to (1) or (2) above. (4) A yeast transformant into which the recombinant vector according to (3) is introduced.
- the promoter of the present invention has a high expression ability of 15 times or more of the TDH3 promoter and 10 times or more of the GAL10 promoter. Use of such a promoter makes it possible to efficiently produce useful substances such as enzymes and pharmaceutical proteins in yeast.
- FIG. 3 shows the results of culturing TDH3 promoter sequence-RPS intron sequence-yCLuc strain and examining the relative expression level of luciferase (RLU / ( ⁇ l ⁇ OD)) in the culture medium.
- GAL10 promoter sequence-RPS25A intron sequence-yCLuc strain and TDH3 promoter sequence-RPS25A intron sequence-yCLuc strain were cultured, and the relative expression level (RLU / ( ⁇ l ⁇ OD)) of luciferase in the culture was examined.
- FIG. 7 is a diagram showing the results of culturing TDH3 promoter sequence-RPS25A promoter sequence having different lengths-yCLuc strains and examining the relative expression level (RLU / ( ⁇ l ⁇ OD)) of luciferase in the culture medium.
- the promoter of the present invention is a promoter for high expression of a target gene in yeast, comprising a yeast TDH3 or GAL10 gene promoter sequence and a yeast ribosomal protein gene intron sequence in sequence.
- the promoter is not particularly limited as long as the intron sequence of the yeast ribosomal protein gene is any one of the following sequences (a) to (c): Is a base sequence of a region that can control the expression of a gene located downstream by binding to RNA polymerase, and includes an enhancer region.
- the sequence of the promoter of the present invention is not a natural product because it is a combination of the promoter sequence of the yeast TDH3 or GAL10 gene and the intron sequence of the yeast ribosomal protein gene as described above. .
- A the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
- B a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
- C a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4;
- the base sequence shown in SEQ ID NO: 1 is the intron sequence of ribosomal protein gene 24A (RPS24A), the base sequence shown in SEQ ID NO: 2 is the intron sequence of ribosomal protein gene 25A (RPS25A), and SEQ ID NO: 3.
- the base sequence shown is the intron sequence of ribosomal protein gene 26A (RPS26A), and the base sequence shown in SEQ ID NO: 4 is the intron sequence of ribosomal protein gene 26B (RPS26B).
- sequence in which one or several bases are deleted, substituted, added or inserted in the above (b) is 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 Examples include sequences in which ⁇ 2, most preferably 1 base has been deleted, substituted, added or inserted.
- sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs: 1 to 4 in (c) is preferably 93% or more, more preferably 95% or more, and still more preferably 98% or more.
- the yeast is not particularly limited, and examples include yeasts belonging to the genus Saccharomyces, Candida, Kluyveromyces, Pichia, and Schizosaccharomyces. Yeast belonging to the genus Saccharomyces can be preferably mentioned, and Saccharomyces cerevisiae can be more preferably mentioned.
- the target gene may be a full-length gene or a part of it depending on the application.
- the origin may be a gene isolated from any organism or an artificial gene produced by genetic engineering.
- high expression of a target gene means, for example, 15 times or more, preferably 35 times or more, more preferably 35 times or more of a target gene arranged downstream of the promoter sequence as compared with the case where a TDH3 promoter sequence is used.
- the target gene is expressed by transforming a transformant obtained by introducing a recombinant polynucleotide operably linked with a secreted luciferase (CLuc) gene downstream of a promoter sequence into Saccharomyces cerevisiae.
- CLuc secreted luciferase
- a medium 1% yeast extract, 2% peptone, 2% glucose
- the yeast TDH3 or GAL10 promoter sequence in the present invention is not limited to the full-length sequence, but is 1 to 10, more preferably 1 to 5, more preferably 1 to 3, and most preferably the full-length sequence. Is a sequence in which 1 to 2, especially 1 base is deleted, substituted, added or inserted, or 90% or more, preferably 93% or more, more preferably 95% or more, more preferably 98% of the full-length sequence. Sequences with% identity or greater are also included.
- the yeast TDH3 promoter sequence or GAL10 promoter sequence can be amplified by PCR using a yeast chromosome or a commercially available vector containing the yeast TDH3 promoter sequence or GAL10 sequence as a template, or these chromosomes and vectors can be used as restriction enzymes. It can be obtained by processing.
- the yeast TDH3 or GAL10 gene promoter sequence and the yeast ribosomal protein gene intron sequence are sequentially provided as follows: the yeast TDH3 or GAL10 promoter sequence downstream of the yeast ribosomal protein gene intron There is no particular limitation as long as a sequence (hereinafter also simply referred to as “RPS intron sequence”) is operably incorporated, and an arbitrary base sequence is inserted between the yeast TDH3 or GAL10 promoter sequence and the yeast RPS intron sequence.
- the length of such an arbitrary base sequence may be 200 base pairs or less, preferably 100 base pairs or less, more preferably 50 base pairs or less, and 0 base pairs, that is, arbitrary The yeast TDH3 or GAL10 promoter sequence, It is most preferred that the the RPS intron sequences are connected directly.
- the vector used for the recombinant vector of the present invention is not particularly limited as long as it contains the promoter of the present invention and can express the target gene operably incorporated downstream of the promoter, and may be linear or cyclic. Those that are capable of autonomous replication or those that can be integrated into a chromosome are preferred, and those that contain a regulatory sequence such as a terminator or a selection marker may be used.
- the yeast transformant into which the recombinant vector of the present invention has been introduced is a yeast transformant into which the recombinant vector of the present invention has been introduced, and the yeast is preferably a yeast of the genus Saccharomyces, S. cerevisiae is more preferred.
- the method for introducing the recombinant vector of the present invention into yeast is not particularly limited, and is a chemical method such as lithium acetate method, lipofection method, calcium phosphate coprecipitation method, liposome method, DEAE dextran method; A known method such as a method using a specific receptor, a biological method such as a cell fusion method, a physical method such as an electroporation method, a microinjection method, a gene gun method, or an ultrasonic gene transfer method. It can be illustrated.
- the target gene product can be efficiently produced by culturing yeast transformants containing the recombinant vector of the present invention.
- a culture method it can be carried out according to a usual method used for culturing yeast.
- the yeast introduced with the recombinant vector of the present invention is Saccharomyces cerevisiae
- it can be cultured under aerobic conditions such as about 30 ° C., about pH 6, and shaking culture or aeration-agitation culture.
- the target gene product can be recovered from the culture broth or crushed yeast.
- Such a recovery method includes a known protein recovery method such as centrifugation, followed by gel filtration, ion exchange, affinity chromatography, etc. A method of collecting can be mentioned.
- RPS intron sequence-yCLuc strain construction (Construction of RPS24Ap (-730 ⁇ + 469) -yCLuc strain) URA3-5 ′ by PCR using URA3-290 (SEQ ID NO: 5) and URA-290c (SEQ ID NO: 6) as primers from the chromosomal DNA of RAK4333 strain (Fukunaga T et al. Yeast 30: 243-253 (2013)) -TDH3p-yCLuc-URA3 was amplified and introduced into ⁇ snt309 strain (Giaever G et al., Nature 418: 387-391 (2002)) to obtain RAK6164 strain.
- yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers.
- ScURA3-5'40-RPS24Ap-yCLuc + 30 and yCLuc-URA3 using these DNA fragments as templates, ScURA3-5'40-ScRPS24A-730 (SEQ ID NO: 7) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5′40-RPS24Ap-yCLuc-URA3. This was introduced into the S.uracerevisiae ⁇ BY4743 strain (Brachmann CB et al., Yeast 14: 115-132 (1998)) at the position of ura3 ⁇ 0 by the following method.
- YPD liquid medium 1% yeast extract, 2% peptone, 2% glucose
- 9 ml of YPD liquid medium was placed in a 15 ml tube, and 1 ml of the culture solution cultured overnight was added thereto, followed by stationary culture at 28 ° C. for 5 hours.
- the mixture was centrifuged at 8,000 rpm at room temperature for 1 minute, the supernatant was discarded, 5 ml of sterilized water was added, the mixture was centrifuged again under the same conditions, the supernatant was discarded, and the cells were suspended in the remaining solution.
- SD-Ura agar medium 0.17% Yeast Nitrogen Base, 0.5% ammonium sulfate, 2% glucose, 0.0024% adenine sulfate , 0.01% L-Histidine HCl, 0.02% L-Leucine, 0.01% L-Lysine HCl, 0.01% L-Methionene, 0.01% L-Tryptphan, 2% agar powder
- the culture was allowed to stand at 28 ° C. for 3 days to obtain transformant colonies.
- ScURA3-5'40-RPS25Ap-yCLuc + 30 and yCLuc-URA3 using these DNA fragments as templates, ScURA3-5'40-ScRPS25A-1598 (SEQ ID NO: 11) and PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS25Ap-yCLuc-URA3.
- This DNA fragment was introduced into S. cerevisiae BY4743 by the method described above.
- a transformant (RAK8424) obtained by introducing ScURA3-5′40-RPS25Ap-yCLuc-URA3 into S. cerevisiae BY4743 strain has the genotype ura3 ⁇ 0 :: RPS25Ap-yCLuc-URA3.
- yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers.
- yCLuc + 1 SEQ ID NO: 9
- URA3-310c SEQ ID NO: 10
- PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Ap-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
- a transformant (RAK8426) obtained by introducing ScURA3-5′40-RPS26Ap-yCLuc-URA3 into S. cerevisiae BY4743 strain has a genotype of ura3 ⁇ 0 :: RPS26Ap-yCLuc-URA3.
- yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers.
- ScURA3-5'40-RPS26Bp-yCLuc + 30 and yCLuc-URA3 using these DNA fragments as templates, ScURA3-5'40-ScRPS26B-1505 (SEQ ID NO: 15) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Bp-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
- a transformant (RAK8428) obtained by introducing ScURA3-5′40-RPS26Bp-yCLuc-URA3 into S. cerevisiae BY4743 strain has a genotype of ura3 ⁇ 0 :: RPS26Bp-yCLuc-URA3.
- PCR was performed with the combinations of primers shown in Table 1 below, and a 40-base upstream sequence of TDH3 coding sequence known as a high-expression promoter (part of TDH3 promoter sequence), an RPS intron sequence, The DNA fragment of yCLuc-URA3, which has, in order, was amplified.
- Each of the amplified DNA fragments was introduced into the RAK5125 strain (MATa ade2 ⁇ 0A ⁇ his3 ⁇ 1 leu2 ⁇ 0 met15 ⁇ 0 ura3 ⁇ 0 :: ScTDH3p-yGLuc-LEU2) by the above method to obtain transformants.
- Each of the obtained transformants has the following genotype.
- ura3 ⁇ 0 :: TDH3p-RPS24A (+ 1_ + 469) -yCLuc-URA3 ura3 ⁇ 0 :: TDH3p-RPS25A (-327_-1) -yCLuc-URA3 ura3 ⁇ 0 :: TDH3p-RPS26A (-378_-1) -yCLuc-URA3 ura3 ⁇ 0 :: TDH3p-RPS26B (-361_-1) -yCLuc-URA3
- TDH3p in these genotypes is a sequence of 698 bases upstream of the TDH3 coding sequence.
- RPS24A (+ 1_ + 469), RPS25A (-327_-1), RPS26A (-378_-1) and RPS26B (-361_-1) are intron sequences of the respective PRS genes.
- a transformant in which the TDH3p-yCLuc-URA3 gene was introduced into the RAK5125 strain was also prepared.
- Such a transformant has a genotype of ura3 ⁇ 0 :: TDH3p-yCLuc-URA3.
- results are shown in FIG. As shown in FIG. 1, compared with the case where only the TDH3 promoter sequence is used, when the TDH3 promoter sequence and the intron sequence of PRS24A are used, 40 times, when the TDH3 promoter sequence and the intron sequence of PRS25A are used. Was found to be 49 times higher, 39 times higher when using the TDH3 promoter sequence and PRS26A intron sequence, and 17 times higher when using the TDH3 promoter sequence and PRS26B intron sequence.
- GAL10 promoter sequence-RPS intron sequence-construction of yCLuc strain (GAL10 promoter sequence-intron sequence of RPS25A-construction of yCLuc strain)
- the mechanism of inducible expression is indispensable for the production of proteins that adversely affect cells.
- the inducible promoter with the highest expression ability is the GAL10 promoter. Therefore, an inducible promoter having higher expression ability than the GAL10 promoter was attempted by connecting an intron sequence of PRS downstream of the GAL10 promoter sequence.
- a promoter in which 511 bp upstream of the GAL10 coding sequence was used as a galactose promoter and an intron sequence (-327 to -1) of RPS25A was connected downstream thereof was constructed on the Saccharomyces cerevisiae chromosome. The construction method is shown below.
- the obtained fusion DNA fragment TDH3p40-yCLuc-LEU2-URA3-3 ' was introduced into the RAK4314 strain and selected on SD-Leu agar medium to obtain a transformant.
- This transformant (RAK4920) has a genotype of ura3 ⁇ 0 :: TDH3p-yCLuc-LEU2.
- PCR was performed using the yeast RAK4920 chromosome having the genotype of ura3 ⁇ 0 :: TDH3p-yCLuc-LEU2 as a template, yCLuc + 21 (SEQ ID NO: 27) and URA3-280c (SEQ ID NO: 21) as primers, and most of yCLuc was upstream.
- the LEU2 marker possessed by was amplified.
- the URA3 marker of RAK10635 was replaced with this LEU2 marker to construct ura3 ⁇ 0 :: ScRPS25A-600-yCLuc-LEU2 (RAK12003).
- Chromosomes were prepared from this yeast strain, and using this as a template, PCR was performed using ScGAL10p-40 (40) -RPS25A-327 (SEQ ID NO: 28) and URA3-280c (SEQ ID NO: 21) as primers, and GAL10p-40 (40) -RPS25A (-327_-1) -yCLuc-LEU2-URA3-3 ′ was amplified.
- This DNA fragment was introduced into the RAK4315 strain (ura3 ⁇ 0 :: GAL10p-URA3) by the above method, and SD-Leu agar medium (0.17% Yeast Nitrogen Base, 0.5% ammonium sulfate, 2% glucose, 0.003% adenine) sulfate, 0.01% Uracil, 0.01% L-Histidine HCl, 0.01% L-Lysine HCl, 0.01% L-Methionene, 0.01% L-Tryptphan, 2% agar powder) Strains were selected to obtain transformants. The obtained transformant has the genotype of ura3 ⁇ 0 :: GAL10p-RPS25A (-327_-1) -yCLuc-LEU2.
- a strain in which GAL10p-yCLuc-LEU2 was inserted into URA3 ⁇ 0 was constructed as follows. PCR using GAL10p40-yCLuc + 1 (SEQ ID NO: 29) and URA3-280c (SEQ ID NO: 21) as primers using the chromosome of RAK12003 as a template, and ScGAL10-40 (40) -yCLuc-LEU2-URA3-3 ' Amplified. This was introduced into RAK4315 (ura3 ⁇ 0 :: GAL10p-URA3), and a transformant was obtained using SD-Leu agar medium. The obtained transformant has a genotype of ura3 ⁇ 0 :: GAL10p-yCLuc-LEU2.
- ScRPS25A (-500 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-450 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-400 to -1) -yCLuc-URA3 DNA
- the fragment was amplified.
- Each DNA fragment was introduced into RAK5125 by the method described above, and ura3 ⁇ 0 :: ScTDH3p-ScRPS25A (-500 to -1) -yCLuc-URA3, ura3 ⁇ 0 :: ScTDH3p-ScRPS25A (-450 to -1) -yCLuc, respectively.
- a transformant having a genotype of -URA3, ura3 ⁇ 0 :: ScTDH3p-ScRPS25A (-400 to -1) -yCLuc-URA3 was obtained.
- the obtained transformant was composed of an RPS25A promoter sequence (RPS25A -500 to -328 (SEQ ID NO: 33) and an intron sequence of RPS25A -327 to -1 (sequence) including introns and different lengths downstream of the TDH3 promoter. No. 2), from -450 to -328 (SEQ ID NO: 34) of RPS25A and -327 to -1 (SEQ ID NO: 2) of RPS25A, or from -400 to -328 (SEQ ID NO: 35) of RPS25A and -327 of RPS25A. -1 (SEQ ID NO: 2)).
- the promoter of the present invention Since the promoter of the present invention has a high expression ability, it can be used in the field of protein production such as enzymes, vaccines and antibodies. It can also be applied to the field of ethanol production from xylose and cellulose. Furthermore, since the yeast's own sequence is used and self-cloning, it is highly safe and can be used in the food field.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention addresses the problem of providing a promoter that yields higher expression than the TDH3 promoter, the GAL10 promoter, or the like. A promoter that is for causing a target gene to be more highly expressed in yeast. The promoter is prepared so as to comprise, in order, a yeast TDH3 or GAL10 promoter sequence and an intron sequence for a yeast ribosomal protein gene, the intron sequence for the yeast ribosomal protein gene being any of sequences (a)-(c): (a) a base sequence represented by SEQ ID NO:1-4; (b) a sequence that is a base sequence represented by SEQ ID NO:1-4 wherein one or several bases have been deleted, added, inserted, or substituted; (c) a sequence that has 90% or more identity with a base sequence represented by SEQ ID NO:1-4.
Description
本発明は、酵母において目的遺伝子を高発現させるためのプロモーターに関し、より詳しくは、酵母において目的遺伝子を高発現させるためのプロモーターであって、酵母のＴＤＨ３又はＧＡＬ１０遺伝子のプロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えたプロモーターに関する。
The present invention relates to a promoter for high expression of a target gene in yeast, and more particularly, a promoter for high expression of a target gene in yeast, comprising a promoter sequence of yeast TDH3 or GAL10 gene, yeast The present invention relates to a promoter which is sequentially provided with an intron sequence of a somal protein gene.
酵母は酵素や医薬品用タンパク質の生産宿主として利用されており、これらタンパク質の生産性を高めるには高発現プロモーターの利用は不可欠である。これまでに酵母においてタンパク質を高発現させるために、様々な高発現プロモーターが利用されている。かかるプロモーターとしては、恒常的高発現のＴＤＨ３遺伝子のプロモーター（以下、「ＴＤＨ３プロモーター」ともいう）、ＰＧＫ１遺伝子のプロモーター、ＡＤＨ１遺伝子のプロモーター、誘導的高発現のＧＡＬ１遺伝子のプロモーター、ＧＡＬ１０遺伝子のプロモーター（以下、「ＧＡＬ１０プロモーター」ともいう）、ＣＵＰ１遺伝子のプロモーター、ＰＨＯ５遺伝子のプロモーターなどがあり、中でも恒常的に発現量の強さが期待できるものとして、ＴＤＨ３遺伝子のプロモーターやＰＧＫ遺伝子のプロモーターなど、解糖系遺伝子のプロモーターが好んで利用されている。
Yeast is used as a production host for enzymes and pharmaceutical proteins, and the use of a high expression promoter is indispensable to increase the productivity of these proteins. To date, various high expression promoters have been used to highly express proteins in yeast. Examples of such a promoter include a constant high expression TDH3 gene promoter (hereinafter also referred to as “TDH3 promoter”), a PGK1 gene promoter, an ADH1 gene promoter, an inducible high expression GAL1 gene promoter, and a GAL10 gene promoter ( (Hereinafter also referred to as “GAL10 promoter”), CUP1 gene promoter, PHO5 gene promoter, etc. Among them, those that can be expected to have a strong expression level are TDH3 gene promoter, PGK gene promoter, etc. Glycogen promoters are preferred.
また、さらなるタンパク質の生産性向上を求めて、例えば、クルイベロマイセス・マルキシアヌス（Kluyveromyces marxianus）の染色体におけるＰＩＲ１遺伝子の上流に位置し、当該ＰＩＲ１遺伝子の発現を制御している領域からなるプロモーターを利用する方法（特許文献１参照）や、ＴＤＨ３遺伝子の開始コドンの５’側上流１５０塩基対よりさらに５’側上流の非翻訳領域と外来の異種遺伝子とから成るＤＮＡ断片を利用する方法（特許文献２参照）や、酵母のピルビン酸デカルボキシラーゼ遺伝子（ＰＤＣ）のプロモーターの制御下に目的遺伝子をゲノムに導入することを特徴とする遺伝子発現方法（特許文献３参照）や、人工的なイントロン様配列によって高発現させる方法（非特許文献１参照）が提案されている。
Further, in order to further improve protein productivity, for example, a promoter that is located upstream of the PIR1 gene in the chromosome of Kluyveromyces marxianus and controls the expression of the PIR1 gene is provided. A method of using (see Patent Document 1), or a method of using a DNA fragment comprising a non-translated region 5 ′ upstream of the 5 ′ upstream 150 base pairs of the start codon of the TDH3 gene and a foreign heterologous gene (patent) 2), a gene expression method characterized by introducing a target gene into the genome under the control of a yeast pyruvate decarboxylase gene (PDC) promoter (see Patent Document 3), an artificial intron-like A method (see Non-Patent Document 1) for highly expressing the sequence has been proposed.
しかしながら、上記プロモーターを用いても、絶対的な発現量は古くから用いられているＴＤＨ３プロモーターやＧＡＬ１０プロモーターなどからほとんど改善されていないのが現状であり、より高い発現能力を有するプロモーターの開発が求められていた。
However, even if the above promoter is used, the absolute expression level is hardly improved from the TDH3 promoter and the GAL10 promoter which have been used for a long time, and the development of a promoter having higher expression ability is required. It was done.
本発明の課題は、ＴＤＨ３プロモーターやＧＡＬ１０プロモーターよりも高発現のプロモーターを提供することにある。
An object of the present invention is to provide a promoter with higher expression than the TDH3 promoter and the GAL10 promoter.
本発明者らは、酵母遺伝子破壊株を用いたゲノムワイド解析から、イントロンに発現能力を高める作用があることに着目するに至った。また、開始コドン上流にイントロンがあれば、構造遺伝子を操作することなく過剰発現可能であると考えた。そこで、開始コドンより上流、又は開始コドン領域近くにイントロンを含むプロモーター配列をゲノム上から探した。その結果、いくつかのリボソーマルタンパク質（以下、「ＲＰＳ」ともいう）をコードする遺伝子であるリボソーマルタンパク質遺伝子のプロモーター配列が高い活性を持つことを明らかにした。
The present inventors came to pay attention to the fact that introns have an effect of enhancing expression ability from genome-wide analysis using yeast gene disruption strains. In addition, if an intron was present upstream of the start codon, it was considered that overexpression was possible without manipulating the structural gene. Therefore, a promoter sequence containing an intron upstream from the start codon or near the start codon region was searched from the genome. As a result, it has been clarified that the promoter sequence of a ribosomal protein gene, which is a gene encoding several ribosomal proteins (hereinafter also referred to as “RPS”), has high activity.
さらに、これらのプロモーター配列のエンハンサー領域を、一般的高発現プロモーターであるＴＤＨ３プロモーター及び誘導型高発現プロモーターであるＧＡＬ１０プロモーターのエンハンサー領域に置換することでより発現能力が高まると考えた。そこで、分泌性ルシフェラーゼ遺伝子をレポーター遺伝子として、リボソーマルタンパク質遺伝子のプロモーターのイントロン配列の上流の様々な位置にＴＤＨ３プロモーター配列を配置して発現を調べたところ、イントロン配列のすぐ上流にＴＤＨ３プロモーター配列を配置した時に最もルシフェラーゼ活性が高いことが明らかとなった。さらにガラクトース誘導性の高発現プロモーターであるＧＡＬ１０プロモーター配列をリボソーマルタンパク質遺伝子プロモーターのイントロン配列の上流に配置したプロモーターではガラクトース誘導的にルシフェラーゼ活性が高いことを見いだし、本発明を完成した。
Furthermore, it was considered that the expression ability was further increased by replacing the enhancer regions of these promoter sequences with the enhancer regions of the general high expression promoter TDH3 promoter and the inducible high expression promoter GAL10 promoter. Therefore, when the TDH3 promoter sequence was placed at various positions upstream of the intron sequence of the promoter of the ribosomal protein gene using the secreted luciferase gene as a reporter gene, the expression was examined. The TDH3 promoter sequence was immediately upstream of the intron sequence. It was revealed that the luciferase activity was the highest when the was placed. Furthermore, the present inventors have found that a galactose-inducible high-expression promoter GAL10 promoter sequence is located upstream of the intron sequence of a ribosomal protein gene promoter and has a high galactose-inducible luciferase activity.
すなわち、本発明は以下に開示されるとおりのものである。
（１）酵母において目的遺伝子を高発現させるためのプロモーターであって、酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えており、前記酵母のリボソーマルタンパク質遺伝子のイントロン配列が、以下に示す（ａ）～（ｃ）のいずれかの配列であることを特徴とするプロモーター。
（ａ）配列番号１～４に示す塩基配列；
（ｂ）配列番号１～４に示す塩基配列において、１又は数個の塩基が欠失、置換、付加、若しくは挿入された配列；
（ｃ）配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列；
（２）酵母が、サッカロマイセス・セレビシエであることを特徴とする上記（１）記載のプロモーター。
（３）上記（１）又は（２）記載のプロモーターを含む組換えベクター。
（４）上記（３）記載の組換えベクターが導入された酵母の形質転換体。 That is, the present invention is as disclosed below.
(1) A promoter for highly expressing a target gene in yeast, comprising a yeast TDH3 or GAL10 promoter sequence and an intron sequence of a yeast ribosomal protein gene in sequence, and the yeast ribosomal A promoter, wherein the intron sequence of the protein gene is any one of the following sequences (a) to (c):
(A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
(C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4;
(2) The promoter according to (1) above, wherein the yeast is Saccharomyces cerevisiae.
(3) A recombinant vector comprising the promoter according to (1) or (2) above.
(4) A yeast transformant into which the recombinant vector according to (3) is introduced.
（１）酵母において目的遺伝子を高発現させるためのプロモーターであって、酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えており、前記酵母のリボソーマルタンパク質遺伝子のイントロン配列が、以下に示す（ａ）～（ｃ）のいずれかの配列であることを特徴とするプロモーター。
（ａ）配列番号１～４に示す塩基配列；
（ｂ）配列番号１～４に示す塩基配列において、１又は数個の塩基が欠失、置換、付加、若しくは挿入された配列；
（ｃ）配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列；
（２）酵母が、サッカロマイセス・セレビシエであることを特徴とする上記（１）記載のプロモーター。
（３）上記（１）又は（２）記載のプロモーターを含む組換えベクター。
（４）上記（３）記載の組換えベクターが導入された酵母の形質転換体。 That is, the present invention is as disclosed below.
(1) A promoter for highly expressing a target gene in yeast, comprising a yeast TDH3 or GAL10 promoter sequence and an intron sequence of a yeast ribosomal protein gene in sequence, and the yeast ribosomal A promoter, wherein the intron sequence of the protein gene is any one of the following sequences (a) to (c):
(A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
(C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4;
(2) The promoter according to (1) above, wherein the yeast is Saccharomyces cerevisiae.
(3) A recombinant vector comprising the promoter according to (1) or (2) above.
(4) A yeast transformant into which the recombinant vector according to (3) is introduced.
本発明のプロモーターは、ＴＤＨ３プロモーターの１５倍以上、ＧＡＬ１０プロモーターの１０倍以上もの高発現能力を有する。かかるプロモーターを用いることで、酵母における酵素や医薬品用タンパク質などの有用物質を効率よく生産することが可能となる。
The promoter of the present invention has a high expression ability of 15 times or more of the TDH3 promoter and 10 times or more of the GAL10 promoter. Use of such a promoter makes it possible to efficiently produce useful substances such as enzymes and pharmaceutical proteins in yeast.
本発明のプロモーターとしては、酵母において目的遺伝子を高発現させるためのプロモーターであって、酵母のＴＤＨ３又はＧＡＬ１０遺伝子のプロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えており、前記酵母のリボソーマルタンパク質遺伝子のイントロン配列が以下に示す（ａ）～（ｃ）のいずれかの配列であることを特徴とするプロモーターであれば特に制限されず、プロモーターとは、目的遺伝子配列の上流に配置され、ＲＮＡポリメラーゼが結合することにより、下流に配置された遺伝子の発現を制御することができる領域の塩基配列を意味し、エンハンサー領域も含まれる。なお、本発明のプロモーターが備える配列は、上述のように酵母のＴＤＨ３又はＧＡＬ１０遺伝子のプロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを組み合わせた配列であることから、自然産物ではない。
（ａ）配列番号１～４に示す塩基配列；
（ｂ）配列番号１～４に示す塩基配列において、１又は数個の塩基が欠失、置換、付加若しくは挿入された配列；
（ｃ）配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列； The promoter of the present invention is a promoter for high expression of a target gene in yeast, comprising a yeast TDH3 or GAL10 gene promoter sequence and a yeast ribosomal protein gene intron sequence in sequence. The promoter is not particularly limited as long as the intron sequence of the yeast ribosomal protein gene is any one of the following sequences (a) to (c): Is a base sequence of a region that can control the expression of a gene located downstream by binding to RNA polymerase, and includes an enhancer region. The sequence of the promoter of the present invention is not a natural product because it is a combination of the promoter sequence of the yeast TDH3 or GAL10 gene and the intron sequence of the yeast ribosomal protein gene as described above. .
(A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
(C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4;
（ａ）配列番号１～４に示す塩基配列；
（ｂ）配列番号１～４に示す塩基配列において、１又は数個の塩基が欠失、置換、付加若しくは挿入された配列；
（ｃ）配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列； The promoter of the present invention is a promoter for high expression of a target gene in yeast, comprising a yeast TDH3 or GAL10 gene promoter sequence and a yeast ribosomal protein gene intron sequence in sequence. The promoter is not particularly limited as long as the intron sequence of the yeast ribosomal protein gene is any one of the following sequences (a) to (c): Is a base sequence of a region that can control the expression of a gene located downstream by binding to RNA polymerase, and includes an enhancer region. The sequence of the promoter of the present invention is not a natural product because it is a combination of the promoter sequence of the yeast TDH3 or GAL10 gene and the intron sequence of the yeast ribosomal protein gene as described above. .
(A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
(C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4;
なお、配列番号１に示す塩基配列は、リボソーマルタンパク質遺伝子２４Ａ（ＲＰＳ２４Ａ）のイントロン配列、配列番号２に示す塩基配列は、リボソーマルタンパク質遺伝子２５Ａ（ＲＰＳ２５Ａ）のイントロン配列、配列番号３に示す塩基配列は、リボソーマルタンパク質遺伝子２６Ａ（ＲＰＳ２６Ａ）のイントロン配列、配列番号４に示す塩基配列は、リボソーマルタンパク質遺伝子２６Ｂ（ＲＰＳ２６Ｂ）のイントロン配列である。
The base sequence shown in SEQ ID NO: 1 is the intron sequence of ribosomal protein gene 24A (RPS24A), the base sequence shown in SEQ ID NO: 2 is the intron sequence of ribosomal protein gene 25A (RPS25A), and SEQ ID NO: 3. The base sequence shown is the intron sequence of ribosomal protein gene 26A (RPS26A), and the base sequence shown in SEQ ID NO: 4 is the intron sequence of ribosomal protein gene 26B (RPS26B).
上記（ｂ）における１又は数個の塩基が欠失、置換、付加若しくは挿入された配列としては、１～１０個、好ましくは１～５個、より好ましくは１～３個、さらに好ましくは１～２個、最も好ましくは１個の塩基が欠失、置換、付加若しくは挿入された配列を挙げることができる。
The sequence in which one or several bases are deleted, substituted, added or inserted in the above (b) is 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 Examples include sequences in which ˜2, most preferably 1 base has been deleted, substituted, added or inserted.
上記（ｃ）における配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列としては、好ましくは９３％以上、より好ましくは９５％以上、さらに好ましくは９８％以上の同一性を有する配列を挙げることができる。
The sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs: 1 to 4 in (c) is preferably 93% or more, more preferably 95% or more, and still more preferably 98% or more. The arrangement | sequence which has can be mentioned.
酵母としては特に制限されず、サッカロマイセス属（Saccharomyces）、キャンディダ属（Candida）、クルイベロマイセス属（Kluyveromyces）ピキア属（Pichia）、及びシゾサッカロマイセス属（Schizosaccharomyces）に属する酵母を挙げることができ、サッカロマイセス属に属する酵母を好適に挙げることができ、サッカロマイセス・セレビシエをより好適に挙げることができる。
The yeast is not particularly limited, and examples include yeasts belonging to the genus Saccharomyces, Candida, Kluyveromyces, Pichia, and Schizosaccharomyces. Yeast belonging to the genus Saccharomyces can be preferably mentioned, and Saccharomyces cerevisiae can be more preferably mentioned.
上記目的遺伝子としては、用途に合わせて全長の遺伝子でも、その一部でもよい。また、その由来はいかなる生物から単離された遺伝子でも、遺伝子工学的に作製された人工的な遺伝子でもよい。
The target gene may be a full-length gene or a part of it depending on the application. The origin may be a gene isolated from any organism or an artificial gene produced by genetic engineering.
本発明において、目的遺伝子を高発現させるとは、例えば、ＴＤＨ３プロモーター配列を用いた場合と比較して、プロモーター配列の下流に配置した目的遺伝子を１５倍以上、好ましくは３５倍以上、より好ましくは４０倍以上発現させることや、ＧＡＬ１０プロモーター配列を用いた場合と比較して、プロモーター配列の下流に配置した目的遺伝子を８倍以上、好ましくは１０倍以上発現させることを挙げることができる。
In the present invention, high expression of a target gene means, for example, 15 times or more, preferably 35 times or more, more preferably 35 times or more of a target gene arranged downstream of the promoter sequence as compared with the case where a TDH3 promoter sequence is used. For example, expression of 40 times or more, or expression of a target gene arranged downstream of the promoter sequence 8 times or more, preferably 10 times or more, compared to the case of using the GAL10 promoter sequence.
目的遺伝子の発現は、たとえば、プロモーター配列の下流に、分泌型のルシフェラーゼ（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％酵母エキス、２％ペプトン、２％グルコース）にて２８℃、１５０ｒｐｍで２４時間培養したときの培養液中のルシフェラーゼの相対発現量（ＲＬＵ／（μｌ・ＯＤ））を測定することによって求めることができる。
For example, the target gene is expressed by transforming a transformant obtained by introducing a recombinant polynucleotide operably linked with a secreted luciferase (CLuc) gene downstream of a promoter sequence into Saccharomyces cerevisiae. Measure the relative expression level of luciferase (RLU / (μl · OD)) in the culture medium when cultured in a medium (1% yeast extract, 2% peptone, 2% glucose) at 28 ° C. and 150 rpm for 24 hours. Can be obtained.
本発明における酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列としては、その全長の配列だけでなく、全長の配列に対して１～１０個、より好ましくは１～５個、さらに好ましくは１～３個、最も好ましくは１～２個、中でも１個の塩基が欠失、置換、付加若しくは挿入された配列や、全長の配列と９０％以上、好ましくは９３％以上、より好ましくは９５％以上、さらに好ましくは９８％以上の同一性を有する配列も含まれる。なお、酵母のＴＤＨ３プロモーター配列又はＧＡＬ１０プロモーター配列は、酵母の染色体や、酵母のＴＤＨ３プロモーター配列又はＧＡＬ１０配列を含有する市販のベクターをテンプレートとしてＰＣＲにより増幅することや、これらの染色体やベクターを制限酵素処理することによって得ることができる。
The yeast TDH3 or GAL10 promoter sequence in the present invention is not limited to the full-length sequence, but is 1 to 10, more preferably 1 to 5, more preferably 1 to 3, and most preferably the full-length sequence. Is a sequence in which 1 to 2, especially 1 base is deleted, substituted, added or inserted, or 90% or more, preferably 93% or more, more preferably 95% or more, more preferably 98% of the full-length sequence. Sequences with% identity or greater are also included. The yeast TDH3 promoter sequence or GAL10 promoter sequence can be amplified by PCR using a yeast chromosome or a commercially available vector containing the yeast TDH3 promoter sequence or GAL10 sequence as a template, or these chromosomes and vectors can be used as restriction enzymes. It can be obtained by processing.
酵母のＴＤＨ３又はＧＡＬ１０遺伝子のプロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えているとは、酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列の下流に、酵母のリボソーマルタンパク質遺伝子のイントロン配列（以下、単に「ＲＰＳイントロン配列」ともいう）が作動可能に組み込んでいれば特に制限されず、酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列と、酵母のＲＰＳイントロン配列との間には任意の塩基配列を含んでいてもよいが、かかる任意の塩基配列の長さとしては、２００塩基対以下、好ましくは１００塩基対以下、より好ましくは５０塩基対以下を挙げることができ、０塩基対、即ち、任意の塩基配列を含まず、酵母のＴＤＨ３又はＧＡＬ１０プロモーター配列と、酵母のＲＰＳイントロン配列とが直接つながっていることが最も好ましい。
The yeast TDH3 or GAL10 gene promoter sequence and the yeast ribosomal protein gene intron sequence are sequentially provided as follows: the yeast TDH3 or GAL10 promoter sequence downstream of the yeast ribosomal protein gene intron There is no particular limitation as long as a sequence (hereinafter also simply referred to as “RPS intron sequence”) is operably incorporated, and an arbitrary base sequence is inserted between the yeast TDH3 or GAL10 promoter sequence and the yeast RPS intron sequence. The length of such an arbitrary base sequence may be 200 base pairs or less, preferably 100 base pairs or less, more preferably 50 base pairs or less, and 0 base pairs, that is, arbitrary The yeast TDH3 or GAL10 promoter sequence, It is most preferred that the the RPS intron sequences are connected directly.
本発明の組換えベクターに用いるベクターとしては、本発明のプロモーターを含み、該プロモーターの下流に作動可能に組み込んだ目的遺伝子を発現できるものであれば特に制限されず、直鎖状でも環状でもよく、自立複製可能であるものや、あるいは染色体中へ組込み可能であるものが好ましく、また、ターミネーターなどの制御配列や選択マーカーを含有しているものを用いてもよい。
The vector used for the recombinant vector of the present invention is not particularly limited as long as it contains the promoter of the present invention and can express the target gene operably incorporated downstream of the promoter, and may be linear or cyclic. Those that are capable of autonomous replication or those that can be integrated into a chromosome are preferred, and those that contain a regulatory sequence such as a terminator or a selection marker may be used.
本発明の組換えベクターが導入された酵母の形質転換体としては、本発明の組換えベクターが導入された酵母の形質転換体であって、酵母としては、サッカロマイセス属の酵母が好ましく、サッカロマイセス・セレビシエがより好ましい。
The yeast transformant into which the recombinant vector of the present invention has been introduced is a yeast transformant into which the recombinant vector of the present invention has been introduced, and the yeast is preferably a yeast of the genus Saccharomyces, S. cerevisiae is more preferred.
本発明の組換えベクターの酵母への導入方法としては、特に制限されず、酢酸リチウム法、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デキストラン法などの化学的方法；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、細胞融合法などの生物学的方法；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などの物理的方法；などの公知の方法を例示することができる。
The method for introducing the recombinant vector of the present invention into yeast is not particularly limited, and is a chemical method such as lithium acetate method, lipofection method, calcium phosphate coprecipitation method, liposome method, DEAE dextran method; A known method such as a method using a specific receptor, a biological method such as a cell fusion method, a physical method such as an electroporation method, a microinjection method, a gene gun method, or an ultrasonic gene transfer method. It can be illustrated.
本発明の組換えベクターを含む酵母の形質転換体を培養することで、目的遺伝子産物を効率よく生産することができる。培養方法としては酵母の培養に用いられる通常の方法に従って行うことができる。例えば、本発明の組換えベクターが導入された酵母がサッカロマイセス・セレビシエの場合は、３０℃前後、ｐＨ６前後、及び、振とう培養又は通気攪拌培養などの好気的条件下で培養することができる。目的遺伝子産物は培養液又は破砕した酵母から回収することができ、かかる回収する方法としては、公知のタンパク質の回収方法、例えば、遠心分離、次いで、ゲルろ過、イオン交換、アフィニティなどのクロマトグラフィーにより回収する方法を挙げることができる。
The target gene product can be efficiently produced by culturing yeast transformants containing the recombinant vector of the present invention. As a culture method, it can be carried out according to a usual method used for culturing yeast. For example, when the yeast introduced with the recombinant vector of the present invention is Saccharomyces cerevisiae, it can be cultured under aerobic conditions such as about 30 ° C., about pH 6, and shaking culture or aeration-agitation culture. . The target gene product can be recovered from the culture broth or crushed yeast. Such a recovery method includes a known protein recovery method such as centrifugation, followed by gel filtration, ion exchange, affinity chromatography, etc. A method of collecting can be mentioned.
サッカロマイセス・セレビシエのゲノム配列から、コーディング配列上流、あるいは開始コドンＡＴＧ直後に配置されたイントロン配列を探索したところ、ＲＰＳ２４Ａ、ＲＰＳ２５Ａ、ＲＰＳ２６Ａ、ＲＰＳ２６Ｂのイントロン配列が見つかった。そこで、ＴＤＨ３プロモーター配列の下流にＲＰＳ２４Ａ、ＲＰＳ２５Ａ、ＲＰＳ２６Ａ、ＲＰＳ２６Ｂのイントロン配列を配置した人工プロモーターを構築し、ＴＤＨ３プロモーター配列の発現能力と比較した。
When an intron sequence located upstream of the coding sequence or immediately after the start codon ATG was searched from the genome sequence of Saccharomyces cerevisiae, intron sequences of RPS24A, RPS25A, RPS26A, and RPS26B were found. Therefore, an artificial promoter in which intron sequences of RPS24A, RPS25A, RPS26A, and RPS26B are arranged downstream of the TDH3 promoter sequence was constructed and compared with the expression ability of the TDH3 promoter sequence.
［ＲＰＳイントロン配列－ｙＣＬｕｃ株の構築］
（RPS24Ap(-730～+469)-yCLuc株の構築）
RAK4333株（Fukunaga T et al. Yeast 30: 243-253 (2013)）の染色体ＤＮＡからURA3-290（配列番号５）とURA-290c（配列番号６）をプライマーに用いてＰＣＲによりURA3-5’-TDH3p-yCLuc-URA3を増幅し、Δsnt309株（Giaever G et al., Nature 418: 387-391 (2002)）に導入しRAK6164株を得た。 [RPS intron sequence-yCLuc strain construction]
(Construction of RPS24Ap (-730 ～ + 469) -yCLuc strain)
URA3-5 ′ by PCR using URA3-290 (SEQ ID NO: 5) and URA-290c (SEQ ID NO: 6) as primers from the chromosomal DNA of RAK4333 strain (Fukunaga T et al. Yeast 30: 243-253 (2013)) -TDH3p-yCLuc-URA3 was amplified and introduced into Δsnt309 strain (Giaever G et al., Nature 418: 387-391 (2002)) to obtain RAK6164 strain.
（RPS24Ap(-730～+469)-yCLuc株の構築）
RAK4333株（Fukunaga T et al. Yeast 30: 243-253 (2013)）の染色体ＤＮＡからURA3-290（配列番号５）とURA-290c（配列番号６）をプライマーに用いてＰＣＲによりURA3-5’-TDH3p-yCLuc-URA3を増幅し、Δsnt309株（Giaever G et al., Nature 418: 387-391 (2002)）に導入しRAK6164株を得た。 [RPS intron sequence-yCLuc strain construction]
(Construction of RPS24Ap (-730 ～ + 469) -yCLuc strain)
URA3-5 ′ by PCR using URA3-290 (SEQ ID NO: 5) and URA-290c (SEQ ID NO: 6) as primers from the chromosomal DNA of RAK4333 strain (Fukunaga T et al. Yeast 30: 243-253 (2013)) -TDH3p-yCLuc-URA3 was amplified and introduced into Δsnt309 strain (Giaever G et al., Nature 418: 387-391 (2002)) to obtain RAK6164 strain.
作製したRAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS24A-730（配列番号７）とyCLuc+30cNoATG-ScRPS24A+469c（配列番号８）を用いて、ＰＣＲによりＲＰＳ２４Ａの－７３０から＋４６９まで（開始コドンＡＴＧのＡの位置を＋１とする）のＤＮＡ断片（ScURA3-5’40-RPS24Ap-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS24Ap-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS24A-730（配列番号７）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS24Ap-yCLuc-URA3を増幅した。これをS. cerevisiae BY4743株（Brachmann CB et al., Yeast 14:115-132 (1998)）のura3Δ0の位置に次の方法で導入した。
Using the prepared chromosomal DNA of RAK6164 as a template, PCR using RURA24 from -730 of RPS24A using ScURA3-5'40-ScRPS24A-730 (SEQ ID NO: 7) and yCLuc + 30cNoATG-ScRPS24A + 469c (SEQ ID NO: 8) as primers A DNA fragment (ScURA3-5′40-RPS24Ap-yCLuc + 30) up to +469 (the position of A in the start codon ATG is set to +1) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments, ScURA3-5'40-RPS24Ap-yCLuc + 30 and yCLuc-URA3, using these DNA fragments as templates, ScURA3-5'40-ScRPS24A-730 (SEQ ID NO: 7) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5′40-RPS24Ap-yCLuc-URA3. This was introduced into the S.uracerevisiae の BY4743 strain (Brachmann CB et al., Yeast 14: 115-132 (1998)) at the position of ura3Δ0 by the following method.
試験管にＹＰＤ液体培地（１％酵母エキス、２％ペプトン、２％グルコース）を２ｍｌ入れ、酵母を植菌し、２８℃で一晩振とう培養した。１５ｍｌチューブに９ｍｌのＹＰＤ液体培地を入れ、そこに１晩培養した培養液１ｍｌを加え、２８℃で５時間静置培養した。次に８、０００ｒｐｍ、室温で１分間遠心し、上澄みを捨て、５ｍｌの滅菌水を加えて、再び同じ条件で遠心し上澄みを捨て、残った液で細胞を懸濁した。形質転換液（５４％ポリエチレングリコール３３５０、１２０ｍＭ酢酸リチウム、７５０μg／ｍｌサケ精巣ＤＮＡ）１３５μｌに、細胞懸濁液６１μｌ、ＰＣＲで増幅したＤＮＡ溶液４μｌを加え十分混合した。これを４２℃の温浴中に４０分間置いた後、１００μｌの滅菌水を加えＳＤ－Ｕｒａ寒天培地（０．１７％Yeast Nitrogen Base、０．５％硫酸アンモニウム、２％グルコース、０．００２４％adenine sulfate、０．０１％L-Histidine HCl、０．０２％L-Leucine、０．０１％L-Lysine HCl、０．０１％L-Methionene、０．０１％L-Tryptphan、２％寒天末）に広げ、２８℃で３日間静置培養し、形質転換体のコロニーを得た。
2 ml of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) was put in a test tube, the yeast was inoculated, and cultured with shaking at 28 ° C. overnight. 9 ml of YPD liquid medium was placed in a 15 ml tube, and 1 ml of the culture solution cultured overnight was added thereto, followed by stationary culture at 28 ° C. for 5 hours. Next, the mixture was centrifuged at 8,000 rpm at room temperature for 1 minute, the supernatant was discarded, 5 ml of sterilized water was added, the mixture was centrifuged again under the same conditions, the supernatant was discarded, and the cells were suspended in the remaining solution. To 135 μl of the transformant (54% polyethylene glycol 3350, 120 mM lithium acetate, 750 μg / ml salmon testis DNA), 61 μl of the cell suspension and 4 μl of the DNA solution amplified by PCR were added and mixed well. After placing this in a 42 ° C. bath for 40 minutes, 100 μl of sterilized water was added, and SD-Ura agar medium (0.17% Yeast Nitrogen Base, 0.5% ammonium sulfate, 2% glucose, 0.0024% adenine sulfate , 0.01% L-Histidine HCl, 0.02% L-Leucine, 0.01% L-Lysine HCl, 0.01% L-Methionene, 0.01% L-Tryptphan, 2% agar powder) The culture was allowed to stand at 28 ° C. for 3 days to obtain transformant colonies.
ScURA3-5’40-RPS24Ap-yCLuc-URA3をS. cerevisiae BY4743株に導入した上記形質転換体（RAK8422）はura3Δ0::RPS24Ap-yCLuc-URA3の遺伝子型を持つ。
The above transformant (RAK8422) in which ScURA3-5′40-RPS24Ap-yCLuc-URA3 was introduced into S. cerevisiae BY4743 has the genotype ura3Δ0 :: RPS24Ap-yCLuc-URA3.
（RPS25Ap(-1598～-1)-yCLuc株の構築）
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS25A-1598（配列番号１１）とyCLuc+30c-ScRPS25A-1c（配列番号１２）を用いて、ＰＣＲによりＲＰＳ２５Ａの－１５９８から－１までのＤＮＡ断片（ScURA3-5’40-RPS25Ap-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS25Ap-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS25A-1598（配列番号１１）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS25Ap-yCLuc-URA3を増幅した。このＤＮＡ断片をS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS25Ap (-1598 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and PCR as primers using ScURA3-5'40-ScRPS25A-1598 (SEQ ID NO: 11) and yCLuc + 30c-ScRPS25A-1c (SEQ ID NO: 12) The DNA fragment (ScURA3-5'40-RPS25Ap-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments, ScURA3-5'40-RPS25Ap-yCLuc + 30 and yCLuc-URA3, using these DNA fragments as templates, ScURA3-5'40-ScRPS25A-1598 (SEQ ID NO: 11) and PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS25Ap-yCLuc-URA3. This DNA fragment was introduced into S. cerevisiae BY4743 by the method described above.
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS25A-1598（配列番号１１）とyCLuc+30c-ScRPS25A-1c（配列番号１２）を用いて、ＰＣＲによりＲＰＳ２５Ａの－１５９８から－１までのＤＮＡ断片（ScURA3-5’40-RPS25Ap-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS25Ap-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS25A-1598（配列番号１１）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS25Ap-yCLuc-URA3を増幅した。このＤＮＡ断片をS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS25Ap (-1598 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and PCR as primers using ScURA3-5'40-ScRPS25A-1598 (SEQ ID NO: 11) and yCLuc + 30c-ScRPS25A-1c (SEQ ID NO: 12) The DNA fragment (ScURA3-5'40-RPS25Ap-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments, ScURA3-5'40-RPS25Ap-yCLuc + 30 and yCLuc-URA3, using these DNA fragments as templates, ScURA3-5'40-ScRPS25A-1598 (SEQ ID NO: 11) and PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS25Ap-yCLuc-URA3. This DNA fragment was introduced into S. cerevisiae BY4743 by the method described above.
ScURA3-5’40-RPS25Ap-yCLuc-URA3をS. cerevisiae BY4743株に導入した形質転換体（RAK8424）はura3Δ0::RPS25Ap-yCLuc-URA3の遺伝子型を持つ。
A transformant (RAK8424) obtained by introducing ScURA3-5′40-RPS25Ap-yCLuc-URA3 into S. cerevisiae BY4743 strain has the genotype ura3Δ0 :: RPS25Ap-yCLuc-URA3.
（RPS26Ap(-1115～-1)-yCLuc株の構築）
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS26A-1115（配列番号１３）とyCLuc+30c-ScRPS26A-1c（配列番号１４）を用いて、ＰＣＲによりＲＰＳ２６Ａの－１１１５から－１までのＤＮＡ断片（ScURA3-5’40-RPS26Ap-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS26Ap-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS26A-1115（配列番号１３）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS26Ap-yCLuc-URA3を増幅した。これをS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS26Ap (-1115 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and primers, ScURA3-5'40-ScRPS26A-1115 (SEQ ID NO: 13) and yCLuc + 30c-ScRPS26A-1c (SEQ ID NO: 14) by PCR, RPS26A -1115 to -1 The DNA fragment (ScURA3-5'40-RPS26Ap-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments of ScURA3-5'40-RPS26Ap-yCLuc + 30 and yCLuc-URA3, these DNA fragments were used as templates, and ScURA3-5'40-ScRPS26A-1115 (SEQ ID NO: 13) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Ap-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS26A-1115（配列番号１３）とyCLuc+30c-ScRPS26A-1c（配列番号１４）を用いて、ＰＣＲによりＲＰＳ２６Ａの－１１１５から－１までのＤＮＡ断片（ScURA3-5’40-RPS26Ap-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS26Ap-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS26A-1115（配列番号１３）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS26Ap-yCLuc-URA3を増幅した。これをS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS26Ap (-1115 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and primers, ScURA3-5'40-ScRPS26A-1115 (SEQ ID NO: 13) and yCLuc + 30c-ScRPS26A-1c (SEQ ID NO: 14) by PCR, RPS26A -1115 to -1 The DNA fragment (ScURA3-5'40-RPS26Ap-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments of ScURA3-5'40-RPS26Ap-yCLuc + 30 and yCLuc-URA3, these DNA fragments were used as templates, and ScURA3-5'40-ScRPS26A-1115 (SEQ ID NO: 13) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Ap-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
ScURA3-5’40-RPS26Ap-yCLuc-URA3をS. cerevisiae BY4743株に導入した形質転換体（RAK8426）はura3Δ0::RPS26Ap-yCLuc-URA3の遺伝子型を持つ。
A transformant (RAK8426) obtained by introducing ScURA3-5′40-RPS26Ap-yCLuc-URA3 into S. cerevisiae BY4743 strain has a genotype of ura3Δ0 :: RPS26Ap-yCLuc-URA3.
（RPS26Bp(-1505～-1)-yCLuc株の構築）
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS26B-1505（配列番号１５）とyCLuc+30c-ScRPS26B-1c（配列番号１６）を用いて、ＰＣＲによりＲＰＳ２６Ｂの－１５０５から－１までのＤＮＡ断片（ScURA3-5’40-RPS26Bp-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS26Bp-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS26B-1505（配列番号１５）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS26Bp-yCLuc-URA3を増幅した。これをS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS26Bp (-1505 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and primers ScURA3-5'40-ScRPS26B-1505 (SEQ ID NO: 15) and yCLuc + 30c-ScRPS26B-1c (SEQ ID NO: 16) by PCR, RPS26B from -1505 to -1 The DNA fragment (ScURA3-5'40-RPS26Bp-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments, ScURA3-5'40-RPS26Bp-yCLuc + 30 and yCLuc-URA3, using these DNA fragments as templates, ScURA3-5'40-ScRPS26B-1505 (SEQ ID NO: 15) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Bp-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
RAK6164の染色体ＤＮＡをテンプレートに、プライマーにScURA3-5’40-ScRPS26B-1505（配列番号１５）とyCLuc+30c-ScRPS26B-1c（配列番号１６）を用いて、ＰＣＲによりＲＰＳ２６Ｂの－１５０５から－１までのＤＮＡ断片（ScURA3-5’40-RPS26Bp-yCLuc+30）を増幅した。同じくRAK6164の染色体ＤＮＡをテンプレートに、yCLuc+1（配列番号９）とURA3-310c（配列番号１０）をプライマーとしてyCLuc-URA3を増幅した。ScURA3-5’40-RPS26Bp-yCLuc+30とyCLuc-URA3の２種のＤＮＡ断片を融合させるために、これらのＤＮＡ断片をテンプレートとし、ScURA3-5’40-ScRPS26B-1505（配列番号１５）とURA3-310c（配列番号１０）をプライマーに用いてＰＣＲを行い、ScURA3-5’40-RPS26Bp-yCLuc-URA3を増幅した。これをS. cerevisiae BY4743株に上記の方法で導入した。 (Construction of RPS26Bp (-1505 ～ -1) -yCLuc strain)
Using RAK6164 chromosomal DNA as a template and primers ScURA3-5'40-ScRPS26B-1505 (SEQ ID NO: 15) and yCLuc + 30c-ScRPS26B-1c (SEQ ID NO: 16) by PCR, RPS26B from -1505 to -1 The DNA fragment (ScURA3-5'40-RPS26Bp-yCLuc + 30) was amplified. Similarly, yCLuc-URA3 was amplified using chromosomal DNA of RAK6164 as a template and yCLuc + 1 (SEQ ID NO: 9) and URA3-310c (SEQ ID NO: 10) as primers. In order to fuse two DNA fragments, ScURA3-5'40-RPS26Bp-yCLuc + 30 and yCLuc-URA3, using these DNA fragments as templates, ScURA3-5'40-ScRPS26B-1505 (SEQ ID NO: 15) PCR was performed using URA3-310c (SEQ ID NO: 10) as a primer to amplify ScURA3-5'40-RPS26Bp-yCLuc-URA3. This was introduced into S. cerevisiae BY4743 strain by the method described above.
ScURA3-5’40-RPS26Bp-yCLuc-URA3をS. cerevisiae BY4743株に導入した形質転換体（RAK8428）はura3Δ0::RPS26Bp-yCLuc-URA3の遺伝子型を持つ。
A transformant (RAK8428) obtained by introducing ScURA3-5′40-RPS26Bp-yCLuc-URA3 into S. cerevisiae BY4743 strain has a genotype of ura3Δ0 :: RPS26Bp-yCLuc-URA3.
［ＴＤＨ３プロモーター配列－ＲＰＳイントロン配列－ｙＣＬｕｃ株の構築］
上記のとおり構築したura3Δ0::RPS24Ap-yCLuc-URA3、ura3Δ0::RPS25Ap-yCLuc-URA3、ura3Δ0::RPS26Ap-yCLuc-URA3、ura3Δ0:: RPS26Bp-yCLuc-URA3のそれぞれの遺伝子型を持つ酵母株から染色体を調製した。これらの染色体をテンプレートとし、下記の表１に示すプライマーの組合せでＰＣＲを行い、高発現プロモーターとして知られているＴＤＨ３コーディング配列上流配列４０塩基（ＴＤＨ３プロモーター配列の一部）と、ＲＰＳイントロン配列とを順次上流に持つyCLuc-URA3のＤＮＡ断片を増幅した。 [Construction of TDH3 promoter sequence-RPS intron sequence-yCLuc strain]
From yeast strains having the genotypes of ura3Δ0 :: RPS24Ap-yCLuc-URA3, ura3Δ0 :: RPS25Ap-yCLuc-URA3, ura3Δ0 :: RPS26Ap-yCLuc-URA3, ura3Δ0 :: RPS26Bp-yCLuc-URA3 constructed as described above Chromosomes were prepared. Using these chromosomes as templates, PCR was performed with the combinations of primers shown in Table 1 below, and a 40-base upstream sequence of TDH3 coding sequence known as a high-expression promoter (part of TDH3 promoter sequence), an RPS intron sequence, The DNA fragment of yCLuc-URA3, which has, in order, was amplified.
上記のとおり構築したura3Δ0::RPS24Ap-yCLuc-URA3、ura3Δ0::RPS25Ap-yCLuc-URA3、ura3Δ0::RPS26Ap-yCLuc-URA3、ura3Δ0:: RPS26Bp-yCLuc-URA3のそれぞれの遺伝子型を持つ酵母株から染色体を調製した。これらの染色体をテンプレートとし、下記の表１に示すプライマーの組合せでＰＣＲを行い、高発現プロモーターとして知られているＴＤＨ３コーディング配列上流配列４０塩基（ＴＤＨ３プロモーター配列の一部）と、ＲＰＳイントロン配列とを順次上流に持つyCLuc-URA3のＤＮＡ断片を増幅した。 [Construction of TDH3 promoter sequence-RPS intron sequence-yCLuc strain]
From yeast strains having the genotypes of ura3Δ0 :: RPS24Ap-yCLuc-URA3, ura3Δ0 :: RPS25Ap-yCLuc-URA3, ura3Δ0 :: RPS26Ap-yCLuc-URA3, ura3Δ0 :: RPS26Bp-yCLuc-URA3 constructed as described above Chromosomes were prepared. Using these chromosomes as templates, PCR was performed with the combinations of primers shown in Table 1 below, and a 40-base upstream sequence of TDH3 coding sequence known as a high-expression promoter (part of TDH3 promoter sequence), an RPS intron sequence, The DNA fragment of yCLuc-URA3, which has, in order, was amplified.
増幅したＤＮＡ断片をそれぞれ、RAK5125株（MATa ade2Δ0AΔhis3Δ1 leu2Δ0 met15Δ0 ura3Δ0::ScTDH3p-yGLuc-LEU2）に上記の方法で導入して形質転換体を得た。得られた形質転換体はそれぞれ下記の遺伝子型を持つ。
ura3Δ0::TDH3p-RPS24A(+1_+469)-yCLuc-URA3
ura3Δ0::TDH3p-RPS25A(-327_-1)-yCLuc-URA3
ura3Δ0::TDH3p-RPS26A(-378_-1)-yCLuc-URA3
ura3Δ0::TDH3p-RPS26B(-361_-1)-yCLuc-URA3 Each of the amplified DNA fragments was introduced into the RAK5125 strain (MATa ade2Δ0AΔhis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 :: ScTDH3p-yGLuc-LEU2) by the above method to obtain transformants. Each of the obtained transformants has the following genotype.
ura3Δ0 :: TDH3p-RPS24A (+ 1_ + 469) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS25A (-327_-1) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS26A (-378_-1) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS26B (-361_-1) -yCLuc-URA3
ura3Δ0::TDH3p-RPS24A(+1_+469)-yCLuc-URA3
ura3Δ0::TDH3p-RPS25A(-327_-1)-yCLuc-URA3
ura3Δ0::TDH3p-RPS26A(-378_-1)-yCLuc-URA3
ura3Δ0::TDH3p-RPS26B(-361_-1)-yCLuc-URA3 Each of the amplified DNA fragments was introduced into the RAK5125 strain (MATa ade2Δ0AΔhis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 :: ScTDH3p-yGLuc-LEU2) by the above method to obtain transformants. Each of the obtained transformants has the following genotype.
ura3Δ0 :: TDH3p-RPS24A (+ 1_ + 469) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS25A (-327_-1) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS26A (-378_-1) -yCLuc-URA3
ura3Δ0 :: TDH3p-RPS26B (-361_-1) -yCLuc-URA3
これらの遺伝子型におけるＴＤＨ３ｐはＴＤＨ３コーディング配列上流６９８塩基の配列である。また、RPS24A(+1_+469)、RPS25A(-327_-1)、RPS26A(-378_-1)及びRPS26B(-361_-1)は、それぞれのＰＲＳ遺伝子のイントロン配列である。
TDH3p in these genotypes is a sequence of 698 bases upstream of the TDH3 coding sequence. RPS24A (+ 1_ + 469), RPS25A (-327_-1), RPS26A (-378_-1) and RPS26B (-361_-1) are intron sequences of the respective PRS genes.
また、コントロールとして、TDH3p-yCLuc-URA3遺伝子をRAK5125株に導入した形質転換体も作製した。かかる形質転換体はura3Δ0::TDH3p-yCLuc-URA3の遺伝子型を持つ。
As a control, a transformant in which the TDH3p-yCLuc-URA3 gene was introduced into the RAK5125 strain was also prepared. Such a transformant has a genotype of ura3Δ0 :: TDH3p-yCLuc-URA3.
［ルシフェラーゼ活性の測定］
２４－ｗｅｌｌマルチウェルプレートの各ウェルにＹＰＤ液体培地を１ｍｌ入れ、上記で作製した形質転換体を植菌し、２８℃、１５０ｒｐｍで２４時間培養した。得られた培養液１０μｌを、ＹＰＤ液体培地１ｍｌを入れた２４－ｗｅｌｌマルチウェルプレートの各ウェルに植菌した。これを２８℃、１５０ｒｐｍで２４時間培養した。培養液の６００ｎｍの濁度（ＯＤ６００）を分光光度計で測定した。ルシフェラーゼ測定キット（アトー社製）とルミノメータ(Glo Max20/20n Luminometer：Promega社製)を用いて培養液中のルシフェラーゼの相対発現量（ＲＬＵ／（μｌ・ＯＤ））をルシフェラーゼ活性として算出することでプロモーターの発現能力を評価した。 [Measurement of luciferase activity]
1 ml of YPD liquid medium was placed in each well of a 24-well multiwell plate, the transformant prepared above was inoculated, and cultured at 28 ° C. and 150 rpm for 24 hours. 10 μl of the obtained culture solution was inoculated into each well of a 24-well multiwell plate containing 1 ml of YPD liquid medium. This was cultured at 28 ° C. and 150 rpm for 24 hours. The turbidity (OD 600 ) at 600 nm of the culture solution was measured with a spectrophotometer. By calculating the relative expression level of luciferase (RLU / (μl · OD)) in the culture solution as luciferase activity using a luciferase measurement kit (manufactured by Ato) and a luminometer (Glo Max20 / 20n Luminometer: manufactured by Promega) The expression ability of the promoter was evaluated.
２４－ｗｅｌｌマルチウェルプレートの各ウェルにＹＰＤ液体培地を１ｍｌ入れ、上記で作製した形質転換体を植菌し、２８℃、１５０ｒｐｍで２４時間培養した。得られた培養液１０μｌを、ＹＰＤ液体培地１ｍｌを入れた２４－ｗｅｌｌマルチウェルプレートの各ウェルに植菌した。これを２８℃、１５０ｒｐｍで２４時間培養した。培養液の６００ｎｍの濁度（ＯＤ６００）を分光光度計で測定した。ルシフェラーゼ測定キット（アトー社製）とルミノメータ(Glo Max20/20n Luminometer：Promega社製)を用いて培養液中のルシフェラーゼの相対発現量（ＲＬＵ／（μｌ・ＯＤ））をルシフェラーゼ活性として算出することでプロモーターの発現能力を評価した。 [Measurement of luciferase activity]
1 ml of YPD liquid medium was placed in each well of a 24-well multiwell plate, the transformant prepared above was inoculated, and cultured at 28 ° C. and 150 rpm for 24 hours. 10 μl of the obtained culture solution was inoculated into each well of a 24-well multiwell plate containing 1 ml of YPD liquid medium. This was cultured at 28 ° C. and 150 rpm for 24 hours. The turbidity (OD 600 ) at 600 nm of the culture solution was measured with a spectrophotometer. By calculating the relative expression level of luciferase (RLU / (μl · OD)) in the culture solution as luciferase activity using a luciferase measurement kit (manufactured by Ato) and a luminometer (Glo Max20 / 20n Luminometer: manufactured by Promega) The expression ability of the promoter was evaluated.
結果を図１に示す。図１に示すように、ＴＤＨ３プロモーター配列のみを用いた場合と比較して、ＴＤＨ３プロモーター配列とＰＲＳ２４Ａのイントロン配列を用いた場合には４０倍、ＴＤＨ３プロモーター配列とＰＲＳ２５Ａのイントロン配列を用いた場合には４９倍、ＴＤＨ３プロモーター配列とＰＲＳ２６Ａのイントロン配列を用いた場合には３９倍、ＴＤＨ３プロモーター配列とＰＲＳ２６Ｂのイントロン配列を用いた場合には１７倍も高い発現能力を有することが明らかとなった。
The results are shown in FIG. As shown in FIG. 1, compared with the case where only the TDH3 promoter sequence is used, when the TDH3 promoter sequence and the intron sequence of PRS24A are used, 40 times, when the TDH3 promoter sequence and the intron sequence of PRS25A are used. Was found to be 49 times higher, 39 times higher when using the TDH3 promoter sequence and PRS26A intron sequence, and 17 times higher when using the TDH3 promoter sequence and PRS26B intron sequence.
［ＧＡＬ１０プロモーター配列－ＲＰＳイントロン配列－ｙＣＬｕｃ株の構築］
（ＧＡＬ１０プロモーター配列－ＲＰＳ２５Ａのイントロン配列－ｙＣＬｕｃ株の構築）
細胞に悪影響を与えるタンパク質の生産では誘導型発現の仕組みは欠かせない。現在、最も発現能力の高い誘導型プロモーターはＧＡＬ１０プロモーターである。そこで、ＧＡＬ１０プロモーター配列の下流にＰＲＳのイントロン配列をつなげることで、ＧＡＬ１０プロモーターよりも発現能力の高い誘導型プロモーターの作製を試みた。具体的には、ＧＡＬ１０コーディング配列上流５１１ｂｐをガラクトースプロモーターとして用い、その下流にＲＰＳ２５Ａのイントロン配列（－３２７～－１）をつなげたプロモーターをサッカロマイセス・セレビシエ染色体上に構築した。以下にその構築方法を示す。 [GAL10 promoter sequence-RPS intron sequence-construction of yCLuc strain]
(GAL10 promoter sequence-intron sequence of RPS25A-construction of yCLuc strain)
The mechanism of inducible expression is indispensable for the production of proteins that adversely affect cells. Currently, the inducible promoter with the highest expression ability is the GAL10 promoter. Therefore, an inducible promoter having higher expression ability than the GAL10 promoter was attempted by connecting an intron sequence of PRS downstream of the GAL10 promoter sequence. Specifically, a promoter in which 511 bp upstream of the GAL10 coding sequence was used as a galactose promoter and an intron sequence (-327 to -1) of RPS25A was connected downstream thereof was constructed on the Saccharomyces cerevisiae chromosome. The construction method is shown below.
（ＧＡＬ１０プロモーター配列－ＲＰＳ２５Ａのイントロン配列－ｙＣＬｕｃ株の構築）
細胞に悪影響を与えるタンパク質の生産では誘導型発現の仕組みは欠かせない。現在、最も発現能力の高い誘導型プロモーターはＧＡＬ１０プロモーターである。そこで、ＧＡＬ１０プロモーター配列の下流にＰＲＳのイントロン配列をつなげることで、ＧＡＬ１０プロモーターよりも発現能力の高い誘導型プロモーターの作製を試みた。具体的には、ＧＡＬ１０コーディング配列上流５１１ｂｐをガラクトースプロモーターとして用い、その下流にＲＰＳ２５Ａのイントロン配列（－３２７～－１）をつなげたプロモーターをサッカロマイセス・セレビシエ染色体上に構築した。以下にその構築方法を示す。 [GAL10 promoter sequence-RPS intron sequence-construction of yCLuc strain]
(GAL10 promoter sequence-intron sequence of RPS25A-construction of yCLuc strain)
The mechanism of inducible expression is indispensable for the production of proteins that adversely affect cells. Currently, the inducible promoter with the highest expression ability is the GAL10 promoter. Therefore, an inducible promoter having higher expression ability than the GAL10 promoter was attempted by connecting an intron sequence of PRS downstream of the GAL10 promoter sequence. Specifically, a promoter in which 511 bp upstream of the GAL10 coding sequence was used as a galactose promoter and an intron sequence (-327 to -1) of RPS25A was connected downstream thereof was constructed on the Saccharomyces cerevisiae chromosome. The construction method is shown below.
ura3Δ0::RPS25Ap-yCLuc-URA3を持つ酵母株（RAK8424)の染色体をテンプレートに、ScURA3-5'40-ScRPS25A-600（配列番号２２）とURA3-280c（配列番号２１）をプライマーに用いてＰＣＲを行い、ScURA3-5'40-ScRPS25A-600-yCLuc-URA3を増幅した。このＤＮＡ断片をRAK3600（Fukunaga T et al. Yeast 30: 243-253 (2013)）へ上記の方法で導入し、形質転換体（RAK10635）を得た。
PCR using the chromosome of yeast strain (RAK8424) with ura3Δ0 :: RPS25Ap-yCLuc-URA3 as template and ScURA3-5'40-ScRPS25A-600 (SEQ ID NO: 22) and URA3-280c (SEQ ID NO: 21) as primers To amplify ScURA3-5'40-ScRPS25A-600-yCLuc-URA3. This DNA fragment was introduced into RAK3600 (Fukunaga T et al. Yeast 30: 243-253 (2013)) by the above method to obtain a transformant (RAK10635).
RAK4296（Fukunaga T et al. Yeast 30: 243-253 (2013)）の染色体ＤＮＡをテンプレートにTDH3p40yCLuc+1（配列番号２３）と15G-yCLuc+1662c（配列番号２４）をプライマーに用いて、ＰＣＲによりTDH3p40-yCLuc-15Cを増幅した。また、RAK3625の染色体DNAをテンプレートに15C-LEU2-1（配列番号２５）とURA3-300c（配列番号２６）をプライマーに用いて、ＰＣＲにより15G-LEU2-URA3-3’を増幅した。これら２つのＤＮＡ断片を１５Ｃ（連続するシトシン１５塩基）配列を介してＰＣＲで融合させた。得られた融合ＤＮＡ断片TDH3p40-yCLuc-LEU2-URA3-3’をRAK4314株に導入しＳＤ－Ｌｅｕ寒天培地で選択し、形質転換体を得た。この形質転換体（RAK4920）はura3Δ0::TDH3p-yCLuc-LEU2の遺伝子型を持つ。
PCR using RAK4296 (Fukunaga T et al. Yeast 30: 243-253 (2013)) as a template and TDH3p40yCLuc + 1 (SEQ ID NO: 23) and 15G-yCLuc + 1662c (SEQ ID NO: 24) as primers TDH3p40-yCLuc-15C was amplified. In addition, 15G-LEU2-URA3-3 'was amplified by PCR using the chromosomal DNA of RAK3625 as a template and 15C-LEU2-1 (SEQ ID NO: 25) and URA3-300c (SEQ ID NO: 26) as primers. These two DNA fragments were fused by PCR through a 15C (continuous cytosine 15 base) sequence. The obtained fusion DNA fragment TDH3p40-yCLuc-LEU2-URA3-3 'was introduced into the RAK4314 strain and selected on SD-Leu agar medium to obtain a transformant. This transformant (RAK4920) has a genotype of ura3Δ0 :: TDH3p-yCLuc-LEU2.
ura3Δ0::TDH3p-yCLuc-LEU2の遺伝子型を持つ酵母RAK4920の染色体をテンプレートに、yCLuc+21（配列番号２７）とURA3-280c（配列番号２１）をプライマーとしてＰＣＲし、yCLucの大部分を上流に持つＬＥＵ２マーカーを増幅した。RAK10635のＵＲＡ３マーカーをこのＬＥＵ２マーカーを用いて置換し、ura3Δ0:: ScRPS25A-600-yCLuc-LEU2を構築した（RAK12003）。この酵母株から染色体を調製し、これをテンプレートとして、ScGAL10p-40(40)-RPS25A-327（配列番号２８）とURA3-280c（配列番号２１）をプライマーに用いてＰＣＲを行い、GAL10p-40(40)-RPS25A(-327_-1)-yCLuc-LEU2-URA3-3’を増幅した。このＤＮＡ断片をRAK4315株（ura3Δ0::GAL10p-URA3）に上記の方法で導入しＳＤ－Ｌｅｕ寒天培地（０．１７％Yeast Nitrogen Base、０．５％硫酸アンモニウム、２％グルコース、０．００３％adenine sulfate、０．０１％Uracil、０．０１％L-Histidine HCl、０．０１％L-Lysine HCl、０．０１％L-Methionene、０．０１％L-Tryptphan、２％寒天末）で増殖した株を選択して形質転換体を得た。得られた形質転換体はura3Δ0::GAL10p-RPS25A(-327_-1)-yCLuc-LEU2の遺伝子型を持つ。
PCR was performed using the yeast RAK4920 chromosome having the genotype of ura3Δ0 :: TDH3p-yCLuc-LEU2 as a template, yCLuc + 21 (SEQ ID NO: 27) and URA3-280c (SEQ ID NO: 21) as primers, and most of yCLuc was upstream. The LEU2 marker possessed by was amplified. The URA3 marker of RAK10635 was replaced with this LEU2 marker to construct ura3Δ0 :: ScRPS25A-600-yCLuc-LEU2 (RAK12003). Chromosomes were prepared from this yeast strain, and using this as a template, PCR was performed using ScGAL10p-40 (40) -RPS25A-327 (SEQ ID NO: 28) and URA3-280c (SEQ ID NO: 21) as primers, and GAL10p-40 (40) -RPS25A (-327_-1) -yCLuc-LEU2-URA3-3 ′ was amplified. This DNA fragment was introduced into the RAK4315 strain (ura3Δ0 :: GAL10p-URA3) by the above method, and SD-Leu agar medium (0.17% Yeast Nitrogen Base, 0.5% ammonium sulfate, 2% glucose, 0.003% adenine) sulfate, 0.01% Uracil, 0.01% L-Histidine HCl, 0.01% L-Lysine HCl, 0.01% L-Methionene, 0.01% L-Tryptphan, 2% agar powder) Strains were selected to obtain transformants. The obtained transformant has the genotype of ura3Δ0 :: GAL10p-RPS25A (-327_-1) -yCLuc-LEU2.
（ＧＡＬ１０プロモーター配列－ｙＣＬｕｃ株の構築）
コントロールとして、GAL10p-yCLuc-LEU2をURA3Δ0に挿入した株を次のように構築した。RAK12003株の染色体をテンプレートにGAL10p40-yCLuc+1（配列番号２９）とURA3-280c（配列番号２１）をプライマーに用いてＰＣＲし、ScGAL10-40(40)-yCLuc -LEU2-URA3-3’を増幅した。これをRAK4315（ura3Δ0::GAL10p-URA3）に導入してSD-Leu寒天培地を用いて形質転換体を得た。得られた形質転換体はura3Δ0::GAL10p-yCLuc-LEU2の遺伝子型を持つ。 (Construction of GAL10 promoter sequence-yCLuc strain)
As a control, a strain in which GAL10p-yCLuc-LEU2 was inserted into URA3Δ0 was constructed as follows. PCR using GAL10p40-yCLuc + 1 (SEQ ID NO: 29) and URA3-280c (SEQ ID NO: 21) as primers using the chromosome of RAK12003 as a template, and ScGAL10-40 (40) -yCLuc-LEU2-URA3-3 ' Amplified. This was introduced into RAK4315 (ura3Δ0 :: GAL10p-URA3), and a transformant was obtained using SD-Leu agar medium. The obtained transformant has a genotype of ura3Δ0 :: GAL10p-yCLuc-LEU2.
コントロールとして、GAL10p-yCLuc-LEU2をURA3Δ0に挿入した株を次のように構築した。RAK12003株の染色体をテンプレートにGAL10p40-yCLuc+1（配列番号２９）とURA3-280c（配列番号２１）をプライマーに用いてＰＣＲし、ScGAL10-40(40)-yCLuc -LEU2-URA3-3’を増幅した。これをRAK4315（ura3Δ0::GAL10p-URA3）に導入してSD-Leu寒天培地を用いて形質転換体を得た。得られた形質転換体はura3Δ0::GAL10p-yCLuc-LEU2の遺伝子型を持つ。 (Construction of GAL10 promoter sequence-yCLuc strain)
As a control, a strain in which GAL10p-yCLuc-LEU2 was inserted into URA3Δ0 was constructed as follows. PCR using GAL10p40-yCLuc + 1 (SEQ ID NO: 29) and URA3-280c (SEQ ID NO: 21) as primers using the chromosome of RAK12003 as a template, and ScGAL10-40 (40) -yCLuc-LEU2-URA3-3 ' Amplified. This was introduced into RAK4315 (ura3Δ0 :: GAL10p-URA3), and a transformant was obtained using SD-Leu agar medium. The obtained transformant has a genotype of ura3Δ0 :: GAL10p-yCLuc-LEU2.
（ルシフェラーゼ活性の測定方法）
２４－ｗｅｌｌマルチウェルプレートの各ウェルにＹＰＤ液体培地を１ｍｌ入れ、上記で作製したura3Δ0::GAL10p-RPS25A(-327_-1)-yCLuc-LEU2、ura3Δ0::GAL10p-yCLuc-LEU2の遺伝子型を持つ形質転換体、及び実施例１で作製したura3Δ0::TDH3p-RPS25A(-327_-1)-yCLuc-URA3、ura3Δ0::TDH3p-yCLuc-URA3の遺伝子型を持つ形質転換体を植菌し、２８℃、１５０ｒｐｍで２４時間培養した。得られた培養液１０μｌを、非誘導条件のＹＰＤ培地又は誘導条件（ガラクトース条件）のＹＰＧａｌ液体培地（１％酵母エキス、２％ペプトン、２％ガラクトース）１ｍｌを入れた２４－ｗｅｌｌマルチウェルプレートの各ウェルに植菌した。これを２８℃、１５０ｒｐｍで２４時間培養した。培養液の６００ｎｍの濁度（ＯＤ600）を分光光度計で測定し、上記と同様の方法でプロモーターの発現能力を評価した。 (Method for measuring luciferase activity)
1 ml of YPD liquid medium is added to each well of a 24-well multiwell plate, and the genotypes of ura3Δ0 :: GAL10p-RPS25A (-327_-1) -yCLuc-LEU2 and ura3Δ0 :: GAL10p-yCLuc-LEU2 prepared above are used. And the transformant having the ura3Δ0 :: TDH3p-RPS25A (-327_-1) -yCLuc-URA3, ura3Δ0 :: TDH3p-yCLuc-URA3 genotype prepared in Example 1, The cells were cultured at 28 ° C. and 150 rpm for 24 hours. 10 μl of the obtained culture broth was added to a 24-well multiwell plate containing 1 ml of non-inducing YPD medium or YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) under induction conditions (galactose conditions). Each well was inoculated. This was cultured at 28 ° C. and 150 rpm for 24 hours. The 600 nm turbidity (OD 600 ) of the culture solution was measured with a spectrophotometer, and the expression ability of the promoter was evaluated in the same manner as described above.
２４－ｗｅｌｌマルチウェルプレートの各ウェルにＹＰＤ液体培地を１ｍｌ入れ、上記で作製したura3Δ0::GAL10p-RPS25A(-327_-1)-yCLuc-LEU2、ura3Δ0::GAL10p-yCLuc-LEU2の遺伝子型を持つ形質転換体、及び実施例１で作製したura3Δ0::TDH3p-RPS25A(-327_-1)-yCLuc-URA3、ura3Δ0::TDH3p-yCLuc-URA3の遺伝子型を持つ形質転換体を植菌し、２８℃、１５０ｒｐｍで２４時間培養した。得られた培養液１０μｌを、非誘導条件のＹＰＤ培地又は誘導条件（ガラクトース条件）のＹＰＧａｌ液体培地（１％酵母エキス、２％ペプトン、２％ガラクトース）１ｍｌを入れた２４－ｗｅｌｌマルチウェルプレートの各ウェルに植菌した。これを２８℃、１５０ｒｐｍで２４時間培養した。培養液の６００ｎｍの濁度（ＯＤ600）を分光光度計で測定し、上記と同様の方法でプロモーターの発現能力を評価した。 (Method for measuring luciferase activity)
1 ml of YPD liquid medium is added to each well of a 24-well multiwell plate, and the genotypes of ura3Δ0 :: GAL10p-RPS25A (-327_-1) -yCLuc-LEU2 and ura3Δ0 :: GAL10p-yCLuc-LEU2 prepared above are used. And the transformant having the ura3Δ0 :: TDH3p-RPS25A (-327_-1) -yCLuc-URA3, ura3Δ0 :: TDH3p-yCLuc-URA3 genotype prepared in Example 1, The cells were cultured at 28 ° C. and 150 rpm for 24 hours. 10 μl of the obtained culture broth was added to a 24-well multiwell plate containing 1 ml of non-inducing YPD medium or YPGal liquid medium (1% yeast extract, 2% peptone, 2% galactose) under induction conditions (galactose conditions). Each well was inoculated. This was cultured at 28 ° C. and 150 rpm for 24 hours. The 600 nm turbidity (OD 600 ) of the culture solution was measured with a spectrophotometer, and the expression ability of the promoter was evaluated in the same manner as described above.
結果を図２に示す。図２に示すように、ガラクトース条件において、ＧＡＬ１０プロモーター配列のみを用いた場合と比較して、ＧＡＬ１０プロモーター配列とＰＲＳ２５Ａのイントロン配列を用いた場合には１１倍も高い発現能力を有することが明らかとなった。さらに、ＧＡＬ１０プロモーターとＰＲＳ２５Ａのイントロン配列を用いた場合でも、グルコース培地では発現が抑制され、ガラクトース培地で発現が１２５５倍向上していた。ＧＡＬ１０プロモーター配列のみを用いた場合の誘導倍率が１５０倍程度であることを考えると、ＧＡＬ１０プロモーター配列とＰＲＳ２５Ａのイントロン配列を用いた場合には非常にガラクトース誘導性が高いことが明らかとなった。
The results are shown in FIG. As shown in FIG. 2, it is clear that the expression ability is 11 times higher when the GAL10 promoter sequence and the PRS25A intron sequence are used in the galactose condition than when only the GAL10 promoter sequence is used. became. Furthermore, even when the GAL10 promoter and the PRS25A intron sequence were used, the expression was suppressed in the glucose medium, and the expression was improved 1255 times in the galactose medium. Considering that the induction ratio when using only the GAL10 promoter sequence is about 150 times, it was revealed that the galactose inducibility is very high when the GAL10 promoter sequence and the intron sequence of PRS25A are used.
さらに、極めて高い発現能力を有するＴＤＨ３プロモーター配列とＰＲＳ２５Ａのイントロン配列を用いた場合と比較しても７０％近くもの発現能力を有しており、ＧＡＬ１０プロモーター配列とＰＲＳ２５Ａのイントロン配列を用いると、誘導性及び発現能力が高いプロモーターとなることが明らかとなった。
Furthermore, it has an expression ability as close to 70% as compared with the case where the TDH3 promoter sequence having an extremely high expression ability and the intron sequence of PRS25A are used, and when the GAL10 promoter sequence and the intron sequence of PRS25A are used, induction It became clear that it becomes a promoter with high sex and expression ability.
［ＴＤＨ３プロモーター配列－長さの異なるＲＰＳ２５Ａプロモーター－ｙＣＬｕｃ株の構築］
RAK10635の染色体ＤＮＡをテンプレートとし、ScTDH3(-40～-1)-RPS25Ap-500（配列番号３０）とURA3-280c（配列番号２１）、ScTDH3p-40(40)-RPS25Ap-450（配列番号３１）とURA3-280c（配列番号２１）、又は、ScTDH3p-40(40)-RPS25Ap-400（配列番号３２）とURA3-280c（配列番号２１）の組合せでＰＣＲを行い、それぞれ、ScTDH3p(40)-ScRPS25A(-500～-1)-yCLuc-URA3、ScTDH3p(40)-ScRPS25A(-450～-1)-yCLuc-URA3、ScTDH3p(40)-ScRPS25A(-400～-1)-yCLuc-URA3のＤＮＡ断片を増幅した。それぞれのＤＮＡ断片をRAK5125に上記記載の方法で導入し、それぞれ、ura3Δ0::ScTDH3p-ScRPS25A(-500～-1)-yCLuc-URA3、ura3Δ0::ScTDH3p-ScRPS25A(-450～-1)-yCLuc-URA3、ura3Δ0::ScTDH3p-ScRPS25A(-400～-1)-yCLuc-URA3の遺伝子型を持つ形質転換体を得た。 [Structure of TDH3 promoter sequence-RPS25A promoter of different length-yCLuc strain]
Using the chromosomal DNA of RAK10635 as a template, ScTDH3 (-40 to -1) -RPS25Ap-500 (SEQ ID NO: 30), URA3-280c (SEQ ID NO: 21), ScTDH3p-40 (40) -RPS25Ap-450 (SEQ ID NO: 31) And URA3-280c (SEQ ID NO: 21), or ScTDH3p-40 (40) -RPS25Ap-400 (SEQ ID NO: 32) and URA3-280c (SEQ ID NO: 21) in combination. ScRPS25A (-500 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-450 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-400 to -1) -yCLuc-URA3 DNA The fragment was amplified. Each DNA fragment was introduced into RAK5125 by the method described above, and ura3Δ0 :: ScTDH3p-ScRPS25A (-500 to -1) -yCLuc-URA3, ura3Δ0 :: ScTDH3p-ScRPS25A (-450 to -1) -yCLuc, respectively. A transformant having a genotype of -URA3, ura3Δ0 :: ScTDH3p-ScRPS25A (-400 to -1) -yCLuc-URA3 was obtained.
RAK10635の染色体ＤＮＡをテンプレートとし、ScTDH3(-40～-1)-RPS25Ap-500（配列番号３０）とURA3-280c（配列番号２１）、ScTDH3p-40(40)-RPS25Ap-450（配列番号３１）とURA3-280c（配列番号２１）、又は、ScTDH3p-40(40)-RPS25Ap-400（配列番号３２）とURA3-280c（配列番号２１）の組合せでＰＣＲを行い、それぞれ、ScTDH3p(40)-ScRPS25A(-500～-1)-yCLuc-URA3、ScTDH3p(40)-ScRPS25A(-450～-1)-yCLuc-URA3、ScTDH3p(40)-ScRPS25A(-400～-1)-yCLuc-URA3のＤＮＡ断片を増幅した。それぞれのＤＮＡ断片をRAK5125に上記記載の方法で導入し、それぞれ、ura3Δ0::ScTDH3p-ScRPS25A(-500～-1)-yCLuc-URA3、ura3Δ0::ScTDH3p-ScRPS25A(-450～-1)-yCLuc-URA3、ura3Δ0::ScTDH3p-ScRPS25A(-400～-1)-yCLuc-URA3の遺伝子型を持つ形質転換体を得た。 [Structure of TDH3 promoter sequence-RPS25A promoter of different length-yCLuc strain]
Using the chromosomal DNA of RAK10635 as a template, ScTDH3 (-40 to -1) -RPS25Ap-500 (SEQ ID NO: 30), URA3-280c (SEQ ID NO: 21), ScTDH3p-40 (40) -RPS25Ap-450 (SEQ ID NO: 31) And URA3-280c (SEQ ID NO: 21), or ScTDH3p-40 (40) -RPS25Ap-400 (SEQ ID NO: 32) and URA3-280c (SEQ ID NO: 21) in combination. ScRPS25A (-500 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-450 to -1) -yCLuc-URA3, ScTDH3p (40) -ScRPS25A (-400 to -1) -yCLuc-URA3 DNA The fragment was amplified. Each DNA fragment was introduced into RAK5125 by the method described above, and ura3Δ0 :: ScTDH3p-ScRPS25A (-500 to -1) -yCLuc-URA3, ura3Δ0 :: ScTDH3p-ScRPS25A (-450 to -1) -yCLuc, respectively. A transformant having a genotype of -URA3, ura3Δ0 :: ScTDH3p-ScRPS25A (-400 to -1) -yCLuc-URA3 was obtained.
得られた形質転換体は、ＴＤＨ３プロモーター下流に、イントロンを含み長さの異なるＲＰＳ２５Ａプロモーター配列（ＲＰＳ２５Ａの－５００から－３２８（配列番号３３）とＲＰＳ２５Ａのイントロン配列である－３２７から－１（配列番号２）、ＲＰＳ２５Ａの－４５０から－３２８（配列番号３４）とＲＰＳ２５Ａの－３２７から－１（配列番号２）、又は、ＲＰＳ２５Ａの－４００から－３２８（配列番号３５）とＲＰＳ２５Ａの－３２７から－１（配列番号２））を持つ。これらの形質転換体、及び実施例１で作製したura3Δ0::TDH3p-yCLuc-URA3の遺伝子型を持つ形質転換体を上記と同様の方法で培養し、ルシフェラーゼ活性測定、及び培養液の濁度測定を行い、発現能力を評価した。
The obtained transformant was composed of an RPS25A promoter sequence (RPS25A -500 to -328 (SEQ ID NO: 33) and an intron sequence of RPS25A -327 to -1 (sequence) including introns and different lengths downstream of the TDH3 promoter. No. 2), from -450 to -328 (SEQ ID NO: 34) of RPS25A and -327 to -1 (SEQ ID NO: 2) of RPS25A, or from -400 to -328 (SEQ ID NO: 35) of RPS25A and -327 of RPS25A. -1 (SEQ ID NO: 2)). These transformants and the transformant having the ura3Δ0 :: TDH3p-yCLuc-URA3 genotype prepared in Example 1 were cultured by the same method as above, luciferase activity measurement, and turbidity measurement of the culture solution And the expression ability was evaluated.
結果を図３に示す。図３に示すように、ＴＤＨ３プロモーター配列と、ＲＰＳ２５Ａイントロン配列の間にＲＰＳ２５Ａイントロン配列の上流の配列を１７３塩基対、１２３塩基対、７３塩基対含む場合においても、ＴＤＨ３プロモーター配列のみを用いた場合と比較して、３７倍、２４倍、２７倍高い発現能力を示すことが明らかとなった。また、ＴＤＨ３プロモーター配列と、ＲＰＳ２５Ａイントロン配列とが直接つながっていれば、ＴＤＨ３プロモーター配列のみを用いた場合と比較して、１０３倍も高い発現能力を示すことが明らかとなった。すなわち、ＴＤＨ３プロモーターとＲＰＳ２５Ａイントロン配列の間にＲＰＳ２５Ａイントロン配列の上流の配列の一部を含んでいても高い発現能力を示すが、ＴＤＨ３プロモーター配列と、ＲＰＳ２５Ａイントロン配列とが直接つながっているほうが、より高い発現能力を有することが明らかとなった。
The results are shown in FIG. As shown in FIG. 3, even when the sequence upstream of the RPS25A intron sequence is 173 base pairs, 123 base pairs, and 73 base pairs between the TDH3 promoter sequence and the RPS25A intron sequence, only the TDH3 promoter sequence is used. It was revealed that the expression ability was 37 times, 24 times, and 27 times higher than In addition, it was revealed that if the TDH3 promoter sequence and the RPS25A intron sequence are directly connected, the expression ability is 103 times higher than when only the TDH3 promoter sequence is used. That is, even if a part of the sequence upstream of the RPS25A intron sequence is included between the TDH3 promoter and the RPS25A intron sequence, high expression ability is shown. However, the TDH3 promoter sequence and the RPS25A intron sequence are more directly connected. It was revealed that it has high expression ability.
本発明のプロモーターは高発現能力を有することから、酵素や、ワクチン、抗体などのタンパク質生産分野において利用可能である。また、キシロースやセルロースからのエタノール生産の分野にも応用することが可能である。さらに、酵母自身の配列を使用しており、セルフクローニングであることから、安全性が高く、食品分野でも利用可能である。
Since the promoter of the present invention has a high expression ability, it can be used in the field of protein production such as enzymes, vaccines and antibodies. It can also be applied to the field of ethanol production from xylose and cellulose. Furthermore, since the yeast's own sequence is used and self-cloning, it is highly safe and can be used in the food field.
Claims (4)
- 酵母において目的遺伝子を高発現させるためのプロモーターであって、酵母のＴＤＨ３又はＧＡＬ１０遺伝子のプロモーター配列と、酵母のリボソーマルタンパク質遺伝子のイントロン配列とを順次備えており、前記酵母のリボソーマルタンパク質遺伝子のイントロン配列が、以下に示す（ａ）～（ｃ）のいずれかの配列であることを特徴とするプロモーター。
（ａ）配列番号１～４に示す塩基配列；
（ｂ）配列番号１～４に示す塩基配列において、１又は数個の塩基が欠失、置換、付加、若しくは挿入された配列；
（ｃ）配列番号１～４に示す塩基配列と９０％以上の同一性を有する配列； A promoter for high expression of a target gene in yeast, comprising a promoter sequence of a yeast TDH3 or GAL10 gene and an intron sequence of a yeast ribosomal protein gene in sequence, the yeast ribosomal protein A promoter characterized in that the intron sequence of a gene is any one of the following sequences (a) to (c):
(A) the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(B) a sequence in which one or several bases are deleted, substituted, added or inserted in the base sequences shown in SEQ ID NOs: 1 to 4;
(C) a sequence having 90% or more identity with the base sequence shown in SEQ ID NOs: 1 to 4; - 酵母が、サッカロマイセス・セレビシエであることを特徴とする請求項１記載のプロモーター。 The promoter according to claim 1, wherein the yeast is Saccharomyces cerevisiae.
- 請求項１又は２記載のプロモーターを含む組換えベクター。 A recombinant vector comprising the promoter according to claim 1 or 2.
- 請求項３記載の組換えベクターが導入された酵母の形質転換体。 A yeast transformant into which the recombinant vector according to claim 3 has been introduced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016539830A JPWO2016021149A1 (en) | 2014-08-04 | 2015-07-29 | High expression promoter |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014-158646 | 2014-08-04 | ||
| JP2014158646 | 2014-08-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2016021149A1 true WO2016021149A1 (en) | 2016-02-11 |
Family
ID=55263445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2015/003793 WO2016021149A1 (en) | 2014-08-04 | 2015-07-29 | High-expression promoter |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPWO2016021149A1 (en) |
| WO (1) | WO2016021149A1 (en) |
-
2015
- 2015-07-29 JP JP2016539830A patent/JPWO2016021149A1/en active Pending
- 2015-07-29 WO PCT/JP2015/003793 patent/WO2016021149A1/en active Application Filing
Non-Patent Citations (6)
| Title |
|---|
| DATABASE GenBank [o] 29 July 2014 (2014-07-29), GOFFEAU A ET AL.: "Definition: TPA_inf: Saccharomyces cerevisiae S288c chromosome V, complete sequence", Database accession no. BK006939 * |
| JUNEAU K ET AL.: "High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 104, 23 January 2007 (2007-01-23), pages 1522 - 1527, Retrieved from the Internet <URL:http://www.pnas.org/content/104/5/1522.long> [retrieved on 20150928] * |
| JUNEAU K ET AL.: "Introns regulate RNA and protein abundance in yeast", GENETICS, vol. 174, 2 July 2006 (2006-07-02), pages 511 - 518, Retrieved from the Internet <URL:http://www.genetics.org/content/174/1/511.long> [retrieved on 20150928] * |
| MASAKI KONDO ET AL.: "Enhanced protein production by using intron sequences in the yeast Saccharomyces cerevisiae", ABSTRACTS OF THE 66TH ANNUAL MEETING OF THE SOCIETY FOR BIOTECHNOLOGY, 5 August 2014 (2014-08-05), Japan, pages 19, Retrieved from the Internet <URL:http://ci.nii.ac.jp/naid/110009906091> [retrieved on 20150925] * |
| MASAKI KONDO ET AL.: "Saccharomyces cerevisiae ni Okeru Intron Hairetsu o Riyo shita Tanpakushitsu no Kohatsugen", 2014 NENDO JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, 5 March 2014 (2014-03-05), Retrieved from the Internet <URL:http://www.jsbba.or.jp/MeetingofJSBBA/2014/MeetingofJSBBA2014.pdf> [retrieved on 20150925] * |
| NIEUWINT RTM ET AL.: "The gene for yeast ribosomal protein S31 contains an intron in the leader sequence", CURRENT GENETICS, vol. 10, 1985, pages 1 - 5 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2016021149A1 (en) | 2017-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tanaka et al. | Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation | |
| Yu et al. | Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi | |
| Milne et al. | Cassettes for PCR‐mediated gene tagging in Candida albicans utilizing nourseothricin resistance | |
| Varela et al. | Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains | |
| Sherwood et al. | Convergent evolution of a fused sexual cycle promotes the haploid lifestyle | |
| KR102126985B1 (en) | Protein synthesis system for kit synthesis in vitro, kit and method for manufacturing the same | |
| Cha-Aim et al. | Fusion PCR via novel overlap sequences | |
| CN102839165A (en) | Gene mutation type recombined protease K and industrialized production method thereof | |
| JP2020528760A (en) | Use in the preparation of novel fusion proteins and the improvement of their protein synthesis | |
| Bolotin-Fukuhara | Thirty years of the HAP2/3/4/5 complex | |
| Mizutani et al. | High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins | |
| Kazemi Seresht et al. | Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers | |
| Cunha et al. | Control by sugar of Saccharomyces cerevisiae flocculation for industrial ethanol production | |
| Tone et al. | A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae | |
| KR20170105079A (en) | Combination of bacterial chaperones positively affecting the physiology of a native or engineered eukaryotic cell | |
| Hernandez et al. | Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae | |
| Tüncher et al. | The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A. nidulans and human | |
| Kaur et al. | Methods for controlled protein depletion to study protein function during meiosis | |
| KR102418515B1 (en) | Tandem DNA Elements Can Improve Protein Synthesis Efficiency | |
| Sylvain et al. | Yeast zinc cluster proteins Dal81 and Uga3 cooperate by targeting common coactivators for transcriptional activation of γ-aminobutyrate responsive genes | |
| de Ruijter et al. | Screening for novel genes of Saccharomyces cerevisiae involved in recombinant antibody production | |
| WO2016021149A1 (en) | High-expression promoter | |
| Kimura et al. | Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species | |
| Wang et al. | Construction of a flocculating yeast for fuel ethanol production | |
| Steinborn et al. | Assessment of Hansenula polymorpha and Arxula adeninivorans-derived rDNA-targeting elements for the design of Arxula adeninivorans expression vectors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15830067 Country of ref document: EP Kind code of ref document: A1 |
|
| ENP | Entry into the national phase |
Ref document number: 2016539830 Country of ref document: JP Kind code of ref document: A |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 15830067 Country of ref document: EP Kind code of ref document: A1 |