WO2014157736A1 - モルティエレラ属微生物内で高発現活性を示すプロモーター - Google Patents
モルティエレラ属微生物内で高発現活性を示すプロモーター Download PDFInfo
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- WO2014157736A1 WO2014157736A1 PCT/JP2014/059698 JP2014059698W WO2014157736A1 WO 2014157736 A1 WO2014157736 A1 WO 2014157736A1 JP 2014059698 W JP2014059698 W JP 2014059698W WO 2014157736 A1 WO2014157736 A1 WO 2014157736A1
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Images
Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- 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
Definitions
- the present invention includes a promoter exhibiting high expression activity in Mortierella microorganism cells, a vector containing the promoter, a non-human transformant having the promoter introduced therein, and a protein using the promoter or transformant, A method for producing lipids or fatty acids is provided.
- Mortierella genus fungi such as Mortierella alpina are known to produce polyunsaturated fatty acids (PUFA) including arachidonic acid, and are industrially particularly useful fungi.
- PUFA polyunsaturated fatty acids
- Patent Document 1 In utilizing such fungi, breeding, that is, improving the genetic properties of useful organisms to more desirable properties (variety improvement) has been made.
- breeding is very important from the viewpoint of improving the production efficiency of useful compounds by microorganisms and reducing the production cost of the compounds.
- a transformation method is used.
- a DNA fragment encoding a protein necessary for obtaining a desired trait is expressed under the control of an appropriate gene promoter, introduced into a useful organism (host) to be bred, and transformed. Get a body group. Thereafter, a desirable variety (stock) is selected from these.
- an appropriate gene promoter is required depending on the species of the host and depending on the property to be modified.
- Many techniques have been reported for transformation methods of filamentous fungi to which Mortierella belongs.
- many enzyme genes involved in lipid synthesis systems have been obtained in relation to the lipid production ability of fungi belonging to the genus Mortierella.
- gene promoters required for introducing these useful enzyme genes into the genus Mortierella and expressing them at a high level.
- the present inventors have succeeded in cloning a promoter of a gene highly expressed in Mortierella alpina (M. alpina), thereby completing the present invention. That is, the present invention provides the following polynucleotides, expression vectors, transformants, and methods for producing proteins, lipids or fatty acids using the polynucleotides and transformants.
- the polynucleotide according to any one selected from the group consisting of the following (a) to (c): (A) a polynucleotide containing any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 28; (B) a promoter in a microbial cell having a base sequence having 90% or more identity to any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 28 and belonging to the genus Mortierella (C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any one of the base sequences selected from the group consisting of SEQ ID NOS: 1 to 28; And a polynucleotide exhibiting promoter activity in a microorganism cell belonging to the genus Mortierella [2]
- the promoter activity is at least 500 nmol / (when a GUS reporter gene is expressed in a
- the target gene can be expressed with high efficiency in the cells of microorganisms belonging to the genus Mortierella.
- FIG. 1 It is a figure which shows the example of the vector for evaluating the promoter of this invention.
- the HisP sequence is used interchangeably with the promoter of the present invention.
- the promoter activity induced by the addition of galactose is shown.
- GY medium 10 ml cultured at 28 ° C., 300 rpm for 5 days.
- GY medium 10 ml cultured at 28 ° C., 300 rpm for 3 days. It is a figure which shows the activity of promoter PP3p and its shortened type promoter. GY medium 10 ml, cultured at 28 ° C., 300 rpm for 10 days. It is a figure which shows the activity of promoter PP6p and its shortened promoter. 5 days of GY medium 10ml, 28 °C, 300rpm It is a figure which shows the activity of promoter HSC82p and its shortened promoter. GY medium 10 ml, cultured at 28 ° C., 300 rpm for 5 days.
- the base sequence shall be described with the 5 'end on the left side and the 3' end on the right side.
- the present inventors have identified the M. The first successful cloning of multiple types of promoter sequences from alpina. The present inventors have also confirmed that proteins expressed by these promoters show their biological activity.
- the promoter of the present invention is PP7p, CIT1p, PP3p, PP2p, PP6ps, HSC82p, SSA2p, GAL10-2p and / or a partial sequence thereof (shortened type). These promoter region sequences and their truncated sequences are shown in the table below.
- the base sequences shown in the above table that is, any one sequence selected from the group consisting of SEQ ID NOs: 1 to 28 are hereinafter collectively referred to as “the promoter sequence of the present invention”.
- the present invention provides the following polynucleotides as promoters that exhibit high expression activity in microbial cells belonging to the genus Mortierella.
- the polynucleotide according to any one selected from the group consisting of the following (a) to (c): (A) a polynucleotide containing the promoter sequence of the present invention; (B) a polynucleotide having a base sequence having 90% or more identity to the promoter sequence of the present invention and exhibiting promoter activity in a microbial cell belonging to the genus Mortierella; and (c) of the present invention
- polynucleotides of the present invention are hereinafter referred to as “polynucleotides of the present invention”.
- “having” the promoter sequence of the present invention means “including” the promoter sequence of the present invention. Therefore, additional sequences other than the promoter sequence of the present invention, such as an enhancer sequence, may be added upstream (5 ′ end side) or downstream (3 ′ end side) of the promoter sequence of the present invention.
- Such additional sequences are 1 to 1000 bp, 1 to 900 bp, 1 to 800 bp, 1 to 700 bp, 1 to 600 bp, 1 to 500 bp, 1 to 400 bp, 1 to 300 bp, between the promoter sequence of the present invention, It may be added via a base sequence of 1 to 200 bp, 1 to 100 bp, 1 to 75 bp, 1 to 50 bp, 1 to 25 bp, 1 to 10 bp, or directly linked to the promoter sequence of the present invention (that is, the promoter of the present invention The number of nucleotide residues intervening between the sequence and the additional sequence may be 0).
- polynucleotide means DNA or RNA.
- polynucleotide hybridizing under stringent conditions means, for example, colony hybridization using as a probe all or part of a polynucleotide comprising a base sequence complementary to the promoter sequence of the present invention.
- a hybridization method for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001,” and “Ausubel, Current Protocol, J Can be used.
- “highly stringent conditions” means, for example, (1) 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C., (2) 0.2 ⁇ SSC, 0 1% SDS, 60 ° C., (3) 0.2 ⁇ SSC, 0.1% SDS, 62 ° C., (4) 0.2 ⁇ SSC, 0.1% SDS, 65 ° C., or (5) 0.1 ⁇ SSC,.
- the conditions are 1% SDS and 65 ° C., but are not limited thereto. Under these conditions, it can be expected that DNA having high sequence identity can be efficiently obtained as the temperature is increased.
- factors affecting the stringency of hybridization include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can select these factors as appropriate. By doing so, it is possible to achieve the same stringency.
- Alkphos Direct Labeling and Detection System (GE Healthcare) can be used, for example.
- incubation with the labeled probe is performed overnight, and then the membrane is washed with a primary washing buffer containing 0.1% (w / v) SDS at 55 ° C. After washing, the hybridized DNA can be detected.
- a commercially available reagent for example, PCR labeling mix (Roche Diagnostics) etc.
- DIG digoxigenin
- hybridization can be detected using a DIG nucleic acid detection kit (Roche Diagnostics).
- the promoter sequence of the present invention when calculated using homologous search software such as FASTA and BLAST using default parameters, the promoter sequence of the present invention and 90% or more, 91% or more, 92% Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.
- the polynucleotide include 4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
- the identity of the base sequence is determined according to FASTA (Science 227 (4693): 1435-1441 (1985)) or the algorithm BLAST (Basic Local Alignment Search Tool) (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993).
- Programs called blastn, blastx, tblastn and tblastx based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).
- the default parameters of each program are used.
- promoter activity means that when a gene sequence encoding a protein (hereinafter referred to as “target gene”) is inserted downstream of the promoter of the present invention, an expression product of the gene is obtained.
- expression product means either or both of RNA (eg, hnRNA, mRNA, siRNA, miRNA, etc.) that is a transcription product of the same gene and protein that is a translation product of the same gene.
- the target gene is inserted within the region of 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 50 bp, 30 bp, 10 bp within 5 bp from the 3 ′ end of the promoter sequence of the present invention. Done to be located.
- the target gene is not particularly limited, but is preferably a gene encoding a protein for which an activity measurement method has been established.
- genes include selection marker genes such as neomycin resistance gene, hygromycin B phosphotransferase gene, and expression reporter genes such as LacZ, GFP (Green Fluorescence Protein) and luciferase genes. It is not limited.
- confirmation of promoter activity is performed by measuring GUS activity using a ⁇ -D-glucuronidase (GUS) gene.
- M. as a host.
- the GUS gene preferably has a codon usage of M.P.
- GUS activity is achieved by expressing the GUS gene in the cells of microorganisms belonging to the genus Mortierella using the promoter sequence of the present invention and reacting the GUS protein recovered from the cells with p-nitrophenyl- ⁇ -D-glucuronide.
- the absorbance of the reaction system at a wavelength of 405 nm can be measured over time, and the measured value can be applied to the following equation.
- GUS activity (nmol / (mg ⁇ min)) 1000 ⁇ [(gradient value of absorbance change graph for each sample) / (gradient value of calibration curve graph)] / [(protein concentration of sample) / 5]
- the GUS gene is generally a GUS gene derived from E. coli (CDS sequence: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30), and the present invention is used in the cells of microorganisms belonging to the genus Mortierella.
- CDS sequence: SEQ ID NO: 29 amino acid sequence: SEQ ID NO: 30
- the use of the codon of the GUS gene derived from E. coli is modified for Mortierella microorganisms. May be used.
- modification of codon usage reference can be made to the GUSm vs GUS alignment shown in FIGS. 12A and 12B.
- the promoter activity is at least 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600 when a GUS reporter gene is expressed in a microbial cell belonging to the genus Mortierella by the method described above.
- GUS protein activity 1800, 2000 nmol / (mg ⁇ min) can be obtained.
- the method for introducing the gene into the host cell is as described later.
- the above-described polynucleotide of the present invention can be obtained by a known genetic engineering technique or a known synthesis technique.
- the present invention also provides, in another embodiment, an expression vector containing the polynucleotide of the present invention (hereinafter “the vector of the present invention”).
- the vector of the present invention is usually (Ii) a promoter of the present invention; and (ii) transcription termination and polyadenylation of RNA molecules, comprising an expression cassette comprising as a component a signal that functions in the host cell.
- the vector thus constructed is introduced into a host cell.
- suitable host cells used in the present invention include lipid producing bacteria, yeast and the like.
- lipid-producing bacteria examples include MYCOTAXON, Vol. XLIV, No. 2, pp. 257-265 (1992) can be used.
- microorganisms belonging to the genus Mortierella such as Mortierella elongata IFO8570, Mortierella excigua (Mortierella exigua) IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina IFO8568, ATCC16266, ATCC322C, 0.524, CBS2235, CBS23535 72, CBS 528.72, Microorganisms belonging to Subgenus Mortierella, such as BS 529.72, CBS 608.70, CBS 754.68, or Mortierella isabellana CBS 194.28, IFO 6336, IFO 7824, IFO 8873, IFO 8874, IFO 8874, IFO 8874 IFO 7884, Mortierella Nana IFO 8190, Mortierella Ramaniana IFO 5426, IFO
- Such vectors include, for example, pDura5 (Appl. Microbiol. Biotechnol., 65, 419-425, (2004)) and pBIG35 (Appl. Environ. Microbiol., (2009), vol. 75, p. 5529-5535).
- PD4 Appl. Environ. Microbiol., November 2000, 66 (11), p. 4655-4661), pDZeo (J. Biosci. Bioeng., December 2005, 100 (6), p. 617-622), pDX Vector (Curr. Genet., 2009, 55 (3), p. 349-356), pBIG3ura5 (Appl. Environ. Microbiol., 2009, 75, p. 5529). 5535) was based on the existing expression vector, such as, but the promoter regions of these vectors can be prepared by replacing the promoter sequence of the present invention, expression vectors comprising the base is not limited thereto.
- a selection marker may be used to confirm whether the vector has been introduced.
- Selection markers include auxotrophic markers (ura5, niaD, trp1), drug resistance markers (hygromycine, zeocin), geneticin resistance gene (G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 (1984), cerulenin resistance gene (fas2m, PDR4) (Minoshiku Koshi et al., Biochemistry, 64, 660, 1992; Hussain et al., Gene, 101, 149, 1991), etc. Is available.
- auxotrophic markers include (1) to (15) below, but are not limited thereto.
- Examples of drug resistance markers include hygromycin (Hygromycin B) resistance gene, bleomycin t (pleomycin) resistance gene, Transformation of filmogenousPunitiveEthymogenous44 J. Punt, Cees A.M.J.J.van den Hondel, Bialophos resistance gene (Avalos, J., Geever, R.F., and Case, M.E. 1989. Bialaphos resist nce as a dominant selectable marker in Neurosporacassa. Curr. Genet. 16: 369-372., Sulfonylurea resistance gene (Zhang, S., Fan, Y., Xia, Y.X, and Ny O.
- lipid producing bacteria As a host cell transformation method, a publicly known method can be used. For example, in the case of lipid producing bacteria, electroporation method (Mackenxie DA et al. Appl. Environ. Microbiol., 66, 4655-4661, 2000), particle delivery method (JP 2005-287403 “Lipid producing bacteria” Or the Agrobacterium method can be used, but is not limited thereto.
- electroporation method Mackenxie DA et al. Appl. Environ. Microbiol., 66, 4655-4661, 2000
- particle delivery method JP 2005-287403 “Lipid producing bacteria”
- Agrobacterium method can be used, but is not limited thereto.
- the present invention also provides a method for producing a protein, lipid or fatty acid using the transformant described above.
- Non-human transformants into which the promoter of the present invention has been introduced hereinafter referred to as “transformants of the present invention”
- transformants of the present invention in particular, transformants prepared using microorganisms belonging to the genus Mortierella as host cells are highly expressed. To do. Therefore, the target protein can be efficiently produced by using the transformant of the present invention.
- a target protein can be expressed from a target gene in a transformant cell by operatively introducing the target gene into the vector of the present invention and culturing a transformant transformed with the vector.
- a cell lysate can be prepared from the transformant and the target protein expressed can be recovered from the lysate according to a known method. The purpose protein recovery For more information about the "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol.3, Cold Spring Harbor Laboratory Press 2001", “Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) "and the like.
- the target gene is not particularly limited, but is preferably a gene encoding a lipid synthase (hereinafter “lipid synthesis gene”), for example, acyl CoA synthase, glycerol-3-phosphate acyltransferase, diacylglycerol acyl group Transferase, fatty acid chain lengthenase, ⁇ 9 fatty acid desaturase gene, ⁇ 12 fatty acid desaturase gene, ⁇ 6 fatty acid desaturase gene, ⁇ 5 fatty acid desaturase gene, ⁇ 4 fatty acid desaturase gene, ⁇ 3 Examples include a gene encoding a fatty acid desaturase gene, a lysophospholipid acyltransferase gene, a phosphatidic acid phosphatase gene, a fatty acid synthase gene, an acetyl CoA carboxylase gene, and an ATP: citrate lyase gene.
- lipid synthesis gene for
- lipid synthesis gene When a lipid synthesis gene is expressed using a lipid-synthesizing cell, for example, a lipid-producing bacterium, as a host, a lipid synthase expressed from the gene synthesizes lipid and / or fatty acid. it can. Therefore, by culturing the transformant of the present invention, lipids and / or fatty acids can be produced with high efficiency.
- a lipid-synthesizing cell for example, a lipid-producing bacterium
- Lipids or fatty acids can be extracted from cells transformed according to the present invention as follows.
- cultured cells are obtained according to conventional methods such as centrifugation and filtration after completion of the culture.
- the cells are thoroughly washed and preferably dried. Drying can be performed by freeze drying, air drying, or the like. If necessary, the dried cells are crushed with dynomill or ultrasonic waves, and then extracted with an organic solvent, preferably under a nitrogen stream.
- an organic solvent preferably under a nitrogen stream.
- Fatty acid-containing lipids can be obtained by distilling off the organic solvent from the extract under reduced pressure.
- the extracted fatty acid may be methyl esterified by the hydrochloric acid methanol method or the like.
- the separation of the fatty acid from the lipid containing the fatty acid is performed by concentration and separation by a conventional method (for example, urea addition method, cooling separation method, column chromatography method, etc.) in the state of mixed fatty acid or mixed fatty acid ester. It can be carried out.
- a conventional method for example, urea addition method, cooling separation method, column chromatography method, etc.
- the alpina 1S-4 strain was inoculated into 100 ml of GY2: 1 medium (2% glucose, 1% yeast extract pH 6.0) and cultured with shaking at 28 ° C. for 2 days. The cells were collected by filtration, and genomic DNA was prepared using DNeasy (QIAGEN). The base sequence of the genomic DNA was determined using Roche 454 GS FLX Standard. At that time, the base sequence of the fragment library was determined for 2 runs, and the base sequence of the mate pair library was determined for 3 runs. By assembling the obtained base sequences, 300 Super Contigs were obtained.
- the alpina 1S-4 strain was inoculated into 100 ml of a medium (1.8% glucose, 1% yeast extract, pH 6.0) and pre-cultured at 28 ° C. for 3 days.
- a medium (1.8% glucose, 1% yeast extract, pH 6.0)
- 5 L medium (1.8% glucose, 1% soybean flour, 0.1% olive oil, 0.01% adecanol, 0.3% KH 2 PO 4 , 0.1 % Na 2 SO 4 , 0.05% CaCl 2 .2H 2 O, 0.05% MgCl 2 .6H 2 O, pH 6.0
- inoculating the whole preculture and 300 rpm, 1 vvm, 26 ° C.
- the culture was aerated and stirred for 8 days under the conditions described above. Glucose equivalent to 2%, 2%, and 1.5% was added on days 1, 2 and 3, respectively. Bacteria were collected at each stage of culture 1, 2, 3, 6, and 8 days, and total RNA was prepared by the guanidinium hydrochloride / CsCl method. CDNA was synthesized by SOLiD TM Total RNA-Seq for Whole Transcriptome Libraries (Applied Biosystems) and sequenced by SOLiD.
- the promoter region of a gene considered to be highly expressed in alpina 1S-4 strain or the promoter region of a homologue of a galactose metabolic gene was cloned as follows.
- primers for amplifying each promoter region by PCR were designed as follows. In the primer sequences shown below, the underlined portion indicates a restriction enzyme recognition site. Primers were designed to add XbaI and SpeI recognition sequences to both ends of the promoter region. However, since only the GAL10-2p has a SpeI recognition sequence in the sequence, it was designed to add an XbaI recognition sequence to both ends. “F” and “R” included in the name of the primer indicate that the primer is a forward primer and a reverse primer, respectively.
- Each promoter region was cloned by PCR using the genome of Mortierella alpina strain 1S-4 as a template. As a polymerase, PrimeSTAR GXL (TaKaRa) was used.
- GUSm promoter evaluation vector GUSm gene (SEQ ID NO: 31) (FIG. 12A and FIG. 12B) in which the codon usage of E. coli-derived GUS gene (SEQ ID NO: 29) was modified for Mortierella microorganisms was used as a reporter gene.
- GUSm was ligated to a plasmid pBIG35 (Appl. Environ. Microbiol., (2009), vol. 75, p. 5529-5535) containing a histone promoter (HisP), which is a constitutive expression promoter, to construct an expression cassette.
- pBIG35 Appl. Environ. Microbiol., (2009), vol. 75, p. 5529-5535
- HisP histone promoter
- the expression cassette was further ligated to uracil-required marker gene (ura5) in tandem to construct a transformation binary vector pBIG35ZhGUSm (FIG. 1).
- the GUSm gene used for the vector has a codon usage frequency of M.M. It is a ⁇ -D-glucuronidase gene artificially synthesized in accordance with alpina.
- Ura5 is an M.I. alpina orotate phosphoribosyltransferase gene. HisP is described in M.M. It is the promoter of the alpina histone H4.1 gene.
- SdhBt is an M.I. It is a terminator of the alpina succinate dehydrogenase gene.
- ColE1 ori is an origin of replication
- NPTII is a kanamycin resistance gene
- TrfA is a gene related to plasmid amplification
- Left border and Right border are repetitive sequences for gene transfer.
- the promoter region cloned as described above was excised with restriction enzymes XbaI and SpeI, or XbaI, and inserted into the vector pBIG35ZhGUSm digested with XbaI and SpeI instead of HisP.
- Agrobacterium (Agrobacterium tumefaciens C58C1) was transformed with each promoter evaluation vector by electroporation, and LB-Mg agar medium (1% tryptone, 0.5% yeast extract, 85 mM NaCl, 0.5 mM MgSO). (4 ⁇ 7H 2 O, 0.5 mM NaOH, 1.5% agar, pH 7.0), and cultured at 28 ° C. for 48 hours. Agrobacterium containing the vector was confirmed by PCR.
- Agrobacterium containing the vector was added to 100 mL MM medium (10 mM K 2 HPO 4 , 10 mM KH 2 PO 4 , 2.5 mM NaCl, 2 mM MgSO 4 .7H 2 O, 0.7 mM CaCl 2 , 9 ⁇ M FeSO 4 .7H 2 O 4 cm (NH 4 ) 2 SO 4 , 10 mM glucose, pH 7.0) at 28 ° C., 120 rpm, shaken for 2 days, centrifuged at 5,800 ⁇ g, fresh IM medium (0.
- Co-culture medium (same composition as IM medium, but 10 mM) mixed with alpina ⁇ ura-3 suspension (10 8 mL ⁇ 1 ) and loaded with nitrocellulose membrane (70 mm diameter; hardened low-ash grade 50, Whatman) (Including 5 mM glucose and 1.5% agar instead of glucose) and cultured at 23 ° C. for 2-5 days.
- the membrane was uracil-free and SC agar medium containing 0.03% Nile blue A (Sigma) (5.0 g Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco), 1.7 g (NH 4 ).
- Extraction of protein from bacterial cells 500 ⁇ L of disruption buffer (100 mM Tris-HCl (PH 8.0), 5 mM 2-mercaptoethanol) is added to the collected bacterial cells, and the TOMY bead shocker is used with 0.1 mm diameter glass beads. And crushed twice at 5000 rpm for 30 sec. Centrifugation was performed at 8000 ⁇ g for 10 minutes, and the collected supernatant was further centrifuged at 20400 ⁇ g for 10 minutes, and the supernatant was collected as a protein solution. Protein concentration was measured and diluted to any concentration with disruption buffer as needed. All the above operations were performed on ice.
- disruption buffer 100 mM Tris-HCl (PH 8.0), 5 mM 2-mercaptoethanol
- GUS activity measurement substrate p-nitrophenyl- ⁇ -D-glucuronide
- pH 8.0 pH 8.0
- 160 ⁇ L of this substrate solution and 40 ⁇ L of protein sample were mixed on a 96-well microtiter plate, and the absorbance at 37 ° C. and 405 nm was measured over time.
- a calibration curve was prepared by measuring the absorbance of 0.05, 0.1, 0.2, 0.5 mM p-nitrophenol, and the GUS activity value of each sample was calculated by the following formula.
- GUS activity (nmol / (mg ⁇ min)) 1000 ⁇ [(gradient value of absorbance change graph for each sample) / (gradient value of calibration curve graph)] / [(protein concentration of sample) / 5]
- the amount (nmol) of 1 mg / mL protein that converts p-nitrophenyl- ⁇ -D-glucuronide into p-nitrophenol per minute is defined as 1 unit.
- GUS activity evaluation strains 30 stable transformants selected for evaluation of each promoter were cultured on a GY agar medium as described above, and GUS activity was measured. Ten strains showing moderate GUS activity among 30 strains were selected.
- the selected strains were cultured with shaking in 10 ml of GY liquid medium or 10 ml of soybean flour medium at 28 ° C. and 300 rpm for 5 days. After completion of the culture, the bacterial cells were collected by filtration, and GUS activity was measured. The average value was evaluated as the activity of the promoter. The result is shown in FIG. The promoters evaluated were higher in promoter activity than HisP and GAPp, which are known Mortierella-derived promoters, in GY medium and / or soybean flour medium.
- the promoter GAL10-2p was evaluated as follows. First, 30 stable transformants were cultured at 28 ° C. for 3 days in SC + gal agar medium (SC agar medium containing 2% galactose instead of 2% glucose), and the GUS activity was measured as described above. Ten strains showing GUS activity were selected. Inoculated into GY liquid medium, and galactose was added to 2% on the 4th or 7th day. The culture conditions were 28 ° C. and 300 rpm. FIG. 3 shows GUS activity from the 2nd day to the 14th day from the start of the culture. The expression of promoter GAL10-2p was induced by the addition of galactose.
- DNA fragments were prepared by cutting the upstream region of each promoter, and the promoter activity was evaluated. In order to obtain DNA fragments, the following primers were prepared for each promoter.
- the underlined portion is a restriction enzyme recognition site.
- a reverse primer PP7p R corresponding to the 3 ′ side of each promoter used in Examples
- PCR was carried out using SpeI, CIT1p R SpeI, PP3p R SpeI, PP2p R SpeI, PP6ps R SpeI, HSC82p R SpeI, SSA2p R SpeI, GAL10-2p R XbaI).
- the obtained DNA fragment was excised with restriction enzymes XbaI and SpeI or XbaI and inserted into a promoter evaluation vector. In the same manner as the method described in the item “Transformation of Mortierella alpina”.
- alpina was transformed and a stable transformant was selected.
- GUS activity was measured in the same manner as in the examples.
- the culture days were 3 days (CIT1p), 5 days (PP7p, PP6p, HSC82p, SSA2p, GAPp) or 10 days (PP3p) depending on the characteristics of each promoter. The results are shown in FIGS.
- the present invention it is possible to highly express a target gene in a lipid-producing bacterium, whereby the target protein, lipid and fatty acid can be efficiently synthesized and recovered.
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Abstract
Description
このような菌類を利用する上で、育種、すなわち有用生物の遺伝的性質をより望ましい性質に改良する(品種改良する)ことがなされている。特に醗酵技術では、微生物による有用化合物の生産効率を向上し、当該化合物の製造コストを低減する等の観点から、育種は非常に重要なものとなっている。
より好ましい性質を持った有用生物を育種するためには、形質転換による方法が利用されている。この場合、目的の形質を獲得するために必要なタンパク質をコードするDNA断片を、適切な遺伝子プロモーターの制御下で発現するようにして、育種しようとする有用生物(宿主)に導入し、形質転換体の集団を得る。その後、この中から望ましい品種(株)を選抜することになる。この際、宿主となる生物種に応じて、また改変しようとする性質に応じて適切な遺伝子プロモーターが必要である。
モルティエレラ属の菌類が属する糸状菌の形質転換方法については、多くの技術が報告されている。また、モルティエレラ属の菌類の脂質生産能力に関連して、脂質合成系に関与する多数の酵素遺伝子が取得されている。しかしながら、これらの有用な酵素遺伝子をモルティエレラ属へ導入し、高いレベルで発現させるために必要となる遺伝子プロモーターについては、これまでほとんど報告がない。
[1] 以下の(a)~(c)よりなる群より選ばれるいずれかに記載のポリヌクレオチド:
(a)配列番号1~28からなる群より選択されるいずれか一つの塩基配列を含有するポリヌクレオチド;
(b)配列番号1~28からなる群より選択されるいずれか一つの塩基配列に対して、90%以上の同一性を有する塩基配列を有し、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド;及び
(c)配列番号1~28からなる群より選択されるいずれか一つの塩基配列と相補的な塩基配列からなる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するポリヌクレオチドであって、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド
[2] 前記プロモーター活性は、モルティエレラ属に属する微生物細胞内でGUSレポーター遺伝子を発現させた場合に少なくとも500nmol/(mg・分)のGUSタンパク質活性が確認されるものである、前記[1]に記載のポリヌクレオチド。
[3] 配列番号1~28からなる群より選択されるいずれか一つの塩基配列を含有する、前記[1]に記載のポリヌクレオチド。
[4] DNAである、前記[1]又は[2]に記載のポリヌクレオチド。
[5] 前記[1]~[4]のいずれかに記載のポリヌクレオチドを含有するベクター。
[6] 前記[1]~[4]のいずれかに記載のポリヌクレオチドが導入された非ヒト形質転換体。
[7] 前記[6]に記載のベクターが導入された非ヒト形質転換体。
[8] 前記形質転換体が脂質生産菌である、前記[7]又は[8]に記載の形質転換体。
[9] 前記脂質生産菌が、モルティエレラ・アルピナ(Mortierella alpina)である、前記[8]に記載の形質転換体。
なお、本明細書において引用した全ての文献、および公開公報、特許公報その他の特許文献は、参照として本明細書に組み込むものとする。また、本明細書は、2013年3月27日に出願された本願優先権主張の基礎となる日本国特許出願(特願2013−066265号)の明細書及び図面に記載の内容を包含する。
本発明者らは、後述の実施例において詳細に記載するように、脂質生産菌であるM.alpinaから複数種類のプロモーター配列をクローニングすることに初めて成功した。また、本発明者らは、これらのプロモーターにより発現されるタンパク質がその生物活性を示すことも確認した。
本発明のプロモーターは、PP7p、CIT1p、PP3p、PP2p、PP6ps、HSC82p、SSA2p、GAL10−2p及び又はこれらの部分配列(短縮型)である。これらのプロモーター領域配列及びその短縮型配列を以下の表に示す。
上記表に示す塩基配列、つまり、配列番号1~28からなる群より選択されるいずれか1つの任意の配列を、以下、「本発明のプロモーター配列」と総称する。
以下の(a)~(c)よりなる群より選ばれるいずれかに記載のポリヌクレオチド:
(a)本発明のプロモーター配列を含有するポリヌクレオチド;
(b)本発明のプロモーター配列に対して、90%以上の同一性を有する塩基配列を有し、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド;及び
(c)本発明のプロモーター配列と相補的な塩基配列からなる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するポリヌクレオチドであって、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド
また、本発明において、本発明のプロモーター配列を「有する」とは、本発明のプロモーター配列を「含む」ことを意味する。従って、本発明のプロモーター配列以外の付加的な配列、例えば、エンハンサー配列等が本発明のプロモーター配列の上流(5’末端側)又は下流(3’末端側)に付加されていてもよい。このような付加的な配列は、本発明のプロモーター配列との間に1~1000bp、1~900bp、1~800bp、1~700bp、1~600bp、1~500bp、1~400bp、1~300bp、1~200bp、1~100bp、1~75bp、1~50bp、1~25bp、1~10bpの塩基配列を介して付加されていてもよく、あるいは本発明のプロモーター配列に直結(つまり本発明のプロモーター配列と付加的な配列との間に介在するヌクレオチド残基数が0)していてもよい。
本明細書中、「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」とは、例えば、本発明のプロモーター配列と相補的な塩基配列からなるポリヌクレオチドの全部又は一部をプローブとして、コロニーハイブリダイゼーション法、プラークハイブリダイゼーション法又はサザンハイブリダイゼーション法などを用いることにより得られるポリヌクレオチドをいう。ハイブリダイゼーションの方法としては、例えば、″Sambrook & Russell,Molecular Cloning:A Laboratory Manual Vol.3,Cold Spring Harbor,Laboratory Press 2001″及び″Ausubel,Current Protocols in Molecular Biology,John Wiley & Sons 1987−1997″などに記載されている方法を利用することができる。
ここで、「発現産物」とは、同遺伝子の転写産物であるRNA(例えば、hnRNA、mRNA、siRNA、miRNAなど)及び同遺伝子の翻訳産物であるタンパク質のいずれか一方又は両方を意味する。
GUS活性は、モルティエレラ属に属する微生物の細胞内で本発明のプロモーター配列を用いてGUS遺伝子を発現させ、前記細胞から回収したGUSタンパク質をp−ニトロフェニル−β−D−グルクロニドと反応させて、反応系の波長405nmの吸光度を経時的に測定し、その測定値を以下の式に当てはめることによって測定することができる。
GUS活性(nmol/(mg・min))=
1000×[(各サンプルにおける吸光度の経時変化グラフの勾配値)/(検量線グラフの勾配値)]/[(サンプルのタンパク濃度)/5]
コドンの使用の改変の例については、図12A及び図12Bに示すGUSm vs GUSアラインメントを参照することができる。
宿主細胞に遺伝子を導入する方法については、後述のとおりである。
本発明はまた、別の実施形態において、本発明のポリヌクレオチドを含有する発現ベクター(以下、「本発明のベクター」)を提供する。
本発明のベクターは、通常、
(i)本発明のプロモーター;及び
(ii)RNA分子の転写終結及びポリアデニル化に関し、宿主細胞内で機能するシグナルを構成要素として含む発現カセット
を含むように構成される。
このように構築されるベクターは、宿主細胞に導入される。本発明において使用される適切な宿主細胞の例としては、脂質生産菌、酵母等が挙げられる。
(1) メチオニン要求性マーカー:met1、met2、met3、met4、met5、met6、met7、met8、met10、met13、met14、met20;
(2) チロシン要求性マーカー:tyr1、イソロイシン;
(3) バリン要求性マーカー:ilv1、ilv2、ilv3、ilv5;
(4) フェニルアラニン要求性マーカー:pha2;
(5) グルタミン酸要求性マーカー:GLU3;
(6) トレオニン要求性マーカーthr1、thr4;
(7) アスパラギン酸要求性マーカー:asp1、asp5;
(8) セリン要求性マーカー:ser1、ser2;
(9) アルギニン要求性マーカー:arg1、arg3、arg4、arg5、arg8、arg9、arg80、arg81、arg82、arg84;
(10) ウラシル要求性マーカー:ura1、ura2、ura3、ura4、ura5、ura6;
(11) アデニン要求性マーカー:ade1、ade2、ade3、ade4、ade5、ade6、ade8、ade9、ade12、ADE15;
(12) リシン要求性マーカー:lys1、lys2、lys4、lys5、lys7、lys9、lys11、lys13、lys14;
(13) トリプトファン要求性マーカー:trp1、trp2、trp3、trp4、trp5;
(14) ロイシン要求性マーカー:leu1、leu2、leu3、leu4、leu5;
(15) ヒスチジン要求性マーカー:his1、his2、his3、his4、his5、his6、his7、his8
本発明はまた、別の実施形態において、上記の形質転換体を用いたタンパク質、脂質又は脂肪酸の製造方法を提供する。
本発明のプロモーターが導入された非ヒト形質転換体(以下、「本発明の形質転換体」)、特に、モルティエレラ属に属する微生物を宿主細胞として作成された形質転換体では目的遺伝子が高発現する。従って、本発明の形質転換体を用いれば、効率的に目的タンパク質を生成することができる。
発現された目的タンパク質は、例えば、形質転換体から細胞溶解物を調製し、同溶解物から公知の方法に沿って回収することができる。目的タンパク質回収の詳細については″Sambrook & Russell,Molecular Cloning:A Laboratory Manual Vol.3,Cold Spring Harbor Laboratory Press 2001″、″Methods in Yeast Genetics、A laboratory manual(Cold Spring Harbor Laboratory Press、Cold Spring Harbor,NY)″等を参照することができる。
M.alpina 1S−4株を100mlのGY2:1培地(2%グルコース、1%酵母エキス pH6.0)に植菌し、28℃で2日間振とう培養した。濾過により菌体を集菌し、DNeasy(QIAGEN)を用いてゲノムDNAを調製した。
上記ゲノムDNAの塩基配列を、Roche 454 GS FLX Standardを用いて決定した。その際、フラグメントライブラリーの塩基配列決定を2ラン分、メイトペアライブラリーの塩基配列決定を3ラン分行った。得られた塩基配列をアッセンブリすることにより、300個のSuper Contigが得られた。
M.alpina 1S−4株を100mlの培地(1.8%グルコース、1%酵母エキス、pH6.0)に植菌し、3日間28℃で前培養した。10L培養槽(Able Co.,東京)に5Lの培地(1.8%グルコース、1%大豆粉、0.1%オリーブ油、0.01%アデカノール、0.3%KH2PO4、0.1% Na2SO4、0.05% CaCl2・2H2O、0.05% MgCl2・6H2O、pH6.0)を入れ、前培養物を全量植菌し、300rpm、1vvm、26℃の条件で8日間通気攪拌培養した。培養1、2、及び3日目に各々2%、2%、及び1.5%相当のグルコースを添加した。培養1、2、3、6、及び8日目の各ステージに菌体を回収し、塩酸グアジニン/CsCl法でtotal RNAを調製した。SOLiDTM Total RNA−Seq for Whole Transcriptome Libraries(アプライドバイオシステムズ)により、cDNAを合成し、SOLiDでシーケンシングを行った。
発現解析の結果より、M.alpina 1S−4株で発現量が多いと考えられる遺伝子のプロモーター領域、または、ガラクトース代謝系遺伝子のホモログのプロモーター領域を以下のとおりクローニングした。
まず、各プロモーター領域をPCRにて増幅するためのプライマーを以下の通り設計した。なお、以下に示すプライマーの塩基配列において下線部は制限酵素認識部位を示す。プライマーはプロモーター領域両端にそれぞれXbaI、SpeI認識配列を付加するよう設計した。ただし、GAL10−2pに限り、配列中にSpeI認識配列が存在するため、両端に共にXbaI認識配列を付加するよう設計した。プライマーの名称に含まれる「F」及び「R」は、そのプライマーがそれぞれフォワードプライマー及びリバースプライマーであることを示す。
プロモーターCIT1p
プロモーターPP3p
プロモーターPP2p
プロモーターPP6ps
プロモーターHSC82p
プロモーターSSA2p
プロモーターGAL10−2p
Mortierella alpina 1S−4株のゲノムを鋳型としてPCRにて各プロモーター領域をクローニングした。ポリメラーゼはPrimeSTAR GXL(TaKaRa)を使用した。
大腸菌由来のGUS遺伝子(配列番号29)のコドンの使用をモルティエレラ属の微生物用に改変したGUSm遺伝子(配列番号31)(図12A及び図12B)をレポーター遺伝子として用いた。
GUSmを恒常的発現プロモーターであるヒストンプロモーター(HisP)を含むプラスミドpBIG35(Appl.Environ.Microbiol.,(2009),vol.75,p.5529−5535)に連結し、発現カセットを構築した。当該発現カセットを、さらに、ウラシル要求性のマーカー遺伝子(ura5)とタンデムに連結させ、形質転換用バイナリーベクターpBIG35ZhGUSmを構築した(図1)。なお、ベクターに使用したGUSm遺伝子はコドン使用頻度をM.alpinaに合わせて人工的に合成したβ−D−グルクロニダーゼ遺伝子である。Ura5は、M.alpinaのオロチン酸ホスホリボシルトランスフェラーゼ遺伝子である。HisPは、M.alpinaのヒストンH4.1遺伝子のプロモーターである。SdhBtは、M.alpinaのコハク酸デヒドロゲナーゼ遺伝子のターミネーターである。ColE1 oriは、複製起点、NPTIIはカナマイシン耐性遺伝子、TrfAはプラスミドの増幅にかかる遺伝子であり、Left borderとRight borderは遺伝子転移のための繰返し配列である。
上述のとおりクローニングしたプロモーター領域を制限酵素XbaIとSpeI、またはXbaIで切り出し、XbaI、SpeI消化したベクターpBIG35ZhGUSmにHisPの代わりに挿入した。
M.alpina 1S−4株より特許文献(WO2005/019437)に記載された方法にしたがって誘導したウラシル要求性株Δura−3を0.05mg/mLウラシル含有Czapek−Dox寒天培地(3%スクロース、0.2% NaNO3、0.1% KH2PO4、0.05% KCl、0.05% MgSO4・7H2O、0.001% FeSO4・7H2O、2%寒天、pH6.0)で培養して得た培養物を集菌し、Miracloth(Calbiochem)でろ過することで、M.alpinaΔura−3の胞子懸濁液を調製した。アグロバクテリウム(Agrobacterium tumefaciens C58C1)に、作製した各プロモーター評価用ベクターをエレクトロポレーションで形質転換し、LB−Mg寒天培地(1%トリプトン、0.5%酵母エキス、85mM NaCl、0.5mM MgSO4・7H2O、0.5mM NaOH、1.5%寒天、pH7.0)上で28℃、48時間培養した。PCR法で当該ベクターを含むアグロバクテリウムを確認した。当該ベクターを有するアグロバクテリウムを100mL MM培地(10mM K2HPO4、10mM KH2PO4、2.5mM NaCl、2mM MgSO4・7H2O、0.7mM CaCl2、9μM FeSO4・7H2O、4mM(NH4)2SO4、10mMグルコース、pH7.0)で28℃、120rpm、2日間振とう培養し、5,800×gで遠心分離し、新鮮なIM培地(MM培地に0.5%グリセロール、200μMアセトシリンゴン、40mM 2−(N−モルホリノ)エタンスルホン酸(MES)を加え、pH5.3に調製)を加えて懸濁液を調製した。当該懸濁液を、8~12時間、28℃、300rpmでOD 660=0.4−3.7になるまで振とう培養した。当該菌懸濁液100μLを、等量の前記M.alpinaΔura−3懸濁液(108mL−1)と混合し、ニトロセルロース膜(直径70mm;hardened low−ash grade 50、Whatman)を載せた共培養培地(IM培地と同様の組成、ただし、10mMグルコースの代わりに5mMグルコース及び1.5%寒天を含む)上に塗布し、23℃で2−5日間培養した。共培養後、当該膜をウラシルフリー、0.03% Nile blue A(Sigma)を含むSC寒天培地(5.0g Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate(Difco)、1.7g(NH4)2SO4、、20gグルコース、20mgアデニン、30mgチロシン、1.0mgメチオニン、2.0mgアルギニン、2.0mgヒスチジン、4.0mgリジン、4.0mgトリプトファン、5.0mgスレオニン、6.0mgイソロイシン、6.0mgロイシン、6.0mgフェニルアラニン、寒天20g/L)に移し、28℃で5日間培養した。視認可能な真菌コロニーからの菌糸を、ウラシルフリーSC培地に移した。新鮮なウラシルフリーSC培地に移す作業を2回おこなうことにより、形質を安定して保持する形質転換体を選抜した。
菌株の培養および回収
GY寒天培地(2%グルコース、1%酵母エキス、1.5%寒天)にて28℃、2日間培養した。培養終了後の菌体は、菌体を寒天ごと削り取って回収した。
回収した菌体に500μL破砕バッファ(100mM Tris−HCl(PH8.0)、5mM 2−メルカプトエタノール)を添加し、0.1mm径ガラスビーズを用いてTOMY製ビーズショッカーにて5000rpm、30sec、2回破砕した。8000×g、10min遠心し、回収した上清をさらに20400×g、10min遠心し、上清をタンパク質溶液として回収した。タンパク質濃度を測定し、必要に応じて破砕バッファで任意の濃度に希釈した。以上の操作は全て氷上で行なった。
基質(p−ニトロフェニル−β−D−グルクロニド)を終濃度1.25mMとなるようアッセイ用バッファ(21.7mM NaH2PO4、33.9mM Na2HPO4、1.11mM EDTA(pH8.0))に溶解した。この基質溶液160μLとタンパク質サンプル40μLとを96−wellマイクロタイタープレート上で混合し、37℃、405nmにおける吸光度を経時的に測定した。0.05、0.1、0.2、0.5mM p−ニトロフェノールの吸光度を測定して検量線を作成し、以下の計算式によって各サンプルのGUS活性値を算出した。
GUS活性(nmol/(mg・min))=
1000×[(各サンプルにおける吸光度の経時変化グラフの勾配値)/(検量線グラフの勾配値)]/[(サンプルのタンパク質濃度)/5]
各プロモーター評価用に選抜した安定形質転換株30株を、上述のとおりGY寒天培地で培養し、GUS活性を測定した。30株中で中程度のGUS活性を示した10株を選抜した。
選抜した株をGY液体培地10ml、または大豆粉培地10mlにて、28℃、300rpmで5日間振とう培養した。培養終了後、菌体をろ過により回収し、GUS活性を測定した。その平均値を当該プロモーターの活性として評価した。その結果を図2に示す。
評価したプロモーターは、GY培地および/または大豆粉培地において、既知のモルティエレラ由来のプロモーターであるHisPやGAPpよりもプロモーター活性が高かった。
培養時間によるプロモーター活性の変化を調べるため、各プロモーターにつき選抜した株をGY液体培地 10ml、28℃にて2日間、5日間、7日間、14日間、振とう培養した。培養終了後、菌体をろ過により回収し、GUS活性を測定した。結果を表に示す。
プロモーターGAL10−2pの評価は以下のとおり行った。
まず、安定形質転換株30株をSC+gal寒天培地(2%グルコースの代わりに2%ガラクトースを含むSC寒天培地)にて28℃、3日間培養し、上述のとおりGUS活性を測定し、中程度のGUS活性を示す株を10株選抜した。GY液体培地に植菌し、4日目または7日目にガラクトースを2%になるように添加した。培養条件は、28℃、300rpmとした。培養開始2日目から14日目までのGUS活性を図3に示す。プロモーターGAL10−2pは、ガラクトースの添加により、発現誘導された。
各プロモーターのプロモーター活性に必要な領域を調べるため、各プロモーターの上流領域を削ったDNA断片を作製し、プロモーター活性を評価した。
DNA断片を得るために、各プロモーターにつき以下のプライマーを作製した。なお、下線部は制限酵素認識部位である。
プロモーターPP7p−D1000増幅用プライマー
プロモーターPP7p−D750増幅用プライマー
プロモーターPP7p−D500増幅用プライマー
プロモーターPP7p−D250増幅用プライマー
CIT1p
プロモーターCIT1p−D1300増幅用プライマー
プロモーターCIT1p−D1000増幅用プライマー
プロモーターCIT1p−D700増幅用プライマー
プロモーターCIT1p−D400増幅用プライマー
PP3p
プロモーターPP3p−D1600増幅用プライマー
プロモーターPP3p−D1200増幅用プライマー
プロモーターPP3p−D800増幅用プライマー
プロモーターPP3p−D400増幅用プライマー
プロモーターPP3p−D200増幅用プライマー
PP2p
プロモーターPP2p−D1200増幅用プライマー
プロモーターPP2p−D800増幅用プライマー
プロモーターPP2p−D400増幅用プライマー
プロモーターPP2p−D200増幅用プライマー
プロモーターPP6ps増幅用プライマー
プロモーターPP6ps−D750増幅用プライマー
プロモーターPP6ps−D500増幅用プライマー
プロモーターPP6ps−D100増幅用プライマー
HSC82p
プロモーターHSC82p−D800増幅用プライマー
プロモーターHSC82p−D600増幅用プライマー
プロモーターHSC82p−D400増幅用プライマー
プロモーターHSC82p−D200増幅用プライマー
SSA2p
プロモーターSSA2p−D850増幅用プライマー
プロモーターSSA2p−D600増幅用プライマー
プロモーターSSA2p−D400増幅用プライマー
プロモーターSSA2p−D200増幅用プライマー
GAL10−2p
プロモーターGAL10−2p−D2000増幅用プライマー
プロモーターGAL10−2p−D1600増幅用プライマー
プロモーターGAL10−2p−D1200増幅用プライマー
プロモーターGAL10−2p−D800増幅用プライマー
プロモーターGAL10−2p−D400増幅用プライマー
項目「モルティエレラ・アルピナの形質転換」に記載の手法と同様にM.alpinaを形質転換し、安定形質転換株を選抜した。実施例と同様にGUS活性を測定した。なお、培養日数は、各プロモーターの特性に応じて3日間(CIT1p)、5日間(PP7p、PP6p、HSC82p、SSA2p、GAPp)、あるいは10日間(PP3p)とした。結果を図4~10に示す。
Claims (9)
- 以下の(a)~(c)よりなる群より選ばれるいずれかに記載のポリヌクレオチド:
(a)配列番号1~28からなる群より選択されるいずれか一つの塩基配列を含有するポリヌクレオチド;
(b)配列番号1~28からなる群より選択されるいずれか一つの塩基配列に対して、90%以上の同一性を有する塩基配列を有し、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド;及び
(c)配列番号1~28からなる群より選択されるいずれか一つの塩基配列と相補的な塩基配列からなる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するポリヌクレオチドであって、かつモルティエレラ属に属する微生物細胞内でプロモーター活性を示すポリヌクレオチド。 - 前記プロモーター活性は、モルティエレラ属に属する微生物細胞内でGUSレポーター遺伝子を発現させた場合に少なくとも500nmol/(mg・分)のGUSタンパク質活性が確認されるものである、請求項1に記載のポリヌクレオチド。
- 配列番号1~28からなる群より選択されるいずれか一つの塩基配列を含有する、請求項1に記載のポリヌクレオチド。
- DNAである、請求項1又は2に記載のポリヌクレオチド。
- 請求項1~4のいずれかに記載のポリヌクレオチドを含有するベクター。
- 請求項1~4のいずれかに記載のポリヌクレオチドが導入された非ヒト形質転換体。
- 請求項5に記載のベクターが導入された非ヒト形質転換体。
- 前記形質転換体が脂質生産菌である、請求項7又は8に記載の形質転換体。
- 前記脂質生産菌が、モルティエレラ・アルピナ(Mortierella alpina)である、請求項8に記載の形質転換体。
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EP2980216A4 (en) | 2016-12-14 |
US10323250B2 (en) | 2019-06-18 |
CN105189744B (zh) | 2018-09-18 |
JP6559308B2 (ja) | 2019-08-14 |
CN105189744A (zh) | 2015-12-23 |
RU2019101182A (ru) | 2019-03-04 |
US20190112613A1 (en) | 2019-04-18 |
KR20150136506A (ko) | 2015-12-07 |
EP2980216A1 (en) | 2016-02-03 |
CA2907622A1 (en) | 2014-10-02 |
RU2019101183A (ru) | 2019-03-04 |
US20180087061A1 (en) | 2018-03-29 |
RU2015145333A (ru) | 2017-05-12 |
RU2019101182A3 (ja) | 2019-09-13 |
JP6392208B2 (ja) | 2018-09-19 |
RU2019101183A3 (ja) | 2019-09-13 |
JP2018198618A (ja) | 2018-12-20 |
BR112015024559A2 (pt) | 2017-10-31 |
AU2014244851A1 (en) | 2015-10-22 |
CA2907622C (en) | 2022-08-16 |
US20160152992A1 (en) | 2016-06-02 |
US9765345B2 (en) | 2017-09-19 |
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US10113173B2 (en) | 2018-10-30 |
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