WO2014109360A1 - Procédé pour la production hautement efficace d'une protéine hétérologue - Google Patents

Procédé pour la production hautement efficace d'une protéine hétérologue Download PDF

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WO2014109360A1
WO2014109360A1 PCT/JP2014/050233 JP2014050233W WO2014109360A1 WO 2014109360 A1 WO2014109360 A1 WO 2014109360A1 JP 2014050233 W JP2014050233 W JP 2014050233W WO 2014109360 A1 WO2014109360 A1 WO 2014109360A1
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seq
base sequence
heterologous protein
protein
expression vector
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PCT/JP2014/050233
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Japanese (ja)
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宏子 北本
渡部　貴志
友岳 森田
基夫 小板橋
吉田　重信
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独立行政法人農業環境技術研究所
独立行政法人産業技術総合研究所
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Priority to JP2014556435A priority Critical patent/JP6413117B2/ja
Publication of WO2014109360A1 publication Critical patent/WO2014109360A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • This study relates to a nucleic acid molecule containing the base sequence of the high expression promoter of the foliar yeast Pseudozyma antarctica, an expression vector containing the nucleic acid molecule, and a highly efficient method for producing a heterologous protein using the expression vector.
  • a means for discharging the produced target protein out of the cells and accumulating it in the medium can be said to be one of effective means for improving the protein production amount. It is also effective to use a promoter sequence or terminator sequence with high gene transcription activity to increase the target protein production rate by increasing the gene transcription rate.
  • a promoter sequence or terminator sequence with high gene transcription activity to increase the target protein production rate by increasing the gene transcription rate.
  • the genus Pseudozyma is a plant basidiomycetous yeast generally found from the surface of leaves and fruits.
  • P. antartcica is found in rice stems and leaves, rice cakes, and brown rice, while P. aphidis, P. rugulosa, and P. tsukubaensis are found in the skins of vegetables and fruits such as pakchoi, mizuna and perilla.
  • P. antarctica tens of thousands to hundreds of thousands of live bacteria are isolated per gram of ⁇ , and when analyzing the raw weight of raw vegetables 10 mg, Pseudozyma yeast is isolated from almost all, Humans have a history of eating Pseudozyma yeast with plant foods.
  • Pseudozyma yeast is known to produce lipase, and P. antartcica lipase (CALA, CALB) has abundant industrial use results such as use in detergents.
  • P. antartcica lipase (CALA, CALB)
  • MELs glycolipid mannosyl erythritol lipids
  • P. tsukubaensis ⁇ ⁇ have a high skin protective effect. It is used as an additive for enhancing the functionality of commercial cosmetics.
  • the yeast of the genus Pseudozyma can be expected as a promising host for producing high secretion of heterologous proteins since it has an excellent ability to produce substances.
  • Pseudozyma yeasts are thought to have less protease secretion than the Ascomycetes fungi, so the produced heterologous proteins can be stably accumulated in the medium.
  • biodegradable mulch is about three times that of polyethylene mulch, but since it is poured and decomposed after use, there is no cost for stripping and disposal, and the unit area required for crop harvesting It is estimated that the cost related to multi-use is almost the same. If the usage increases in the future, the price can be expected to fall.
  • Powerful biodegradable plastic-degrading bacteria such as the selected filamentous fungus NITE ⁇ P-573 (Patent Document 2) were selected, biodegradable plastic-degrading enzyme PaE derived from Pseudozyma antarctica, biodegradable plastic derived from filamentous fungus NITE P-573 Enzymes that efficiently decompose biodegradable plastic materials, such as the degrading enzyme PCLE, have been isolated and identified.
  • the degrading enzyme PCLE by applying a PaE or PCLE solution to the surface of the biodegradable mulch, superior degradation characteristics with respect to the biodegradable mulch have been found.
  • Enzymes produced by gene recombination technology are widely used for processing fibers and additives for detergents. Upon introduction, it was confirmed that there was no problem even if it was irritating, allergenic, or discharged into the environment. In addition, based on the safety examination standards of the Ministry of Health, Labor and Welfare, the safety assessment standard for food additives manufactured using genetically modified microorganisms established by the Food Safety Committee of the Cabinet Office (decided on March 25, 2004) ) In principle, genetically modified additives that can be evaluated for safety are limited to the use of recombinants derived from non-pathogenic hosts with experience in the manufacture of additives or food experience. Has been. Enzymes that are applied directly to agricultural mulches after crop harvesting are not directly ingested, but are most safe and suitable for enzymes produced in an experienced host such as Pseudozyma yeast. Therefore, its mass production technology is required.
  • Non-patent Document 4 which induces expression of heterologous protein genes downstream of the promoter in the presence of vegetable oil, maltose, and xylose, respectively.
  • the present invention developed a high-expression promoter of P. antarctica and constructed a heterologous protein production system using the obtained high-expression promoter, and the enzyme was efficiently used using the Ustilaginales (Krochomycetes) strain containing Pseudozyma yeast. It is an object of the present invention to provide a manufacturing method.
  • the present inventors have cultivated foliar yeast P. antarctica using various carbon sources, and as a result, when xylose, which is a constituent of herbaceous biomass, is used as a carbon source, We discovered for the first time that there are unknown proteins produced in large quantities. Further investigation was conducted, and the gene sequence (PANT_8c00118) of this unknown protein was identified from the whole genome sequence of the P. antarctica T34 strain. Furthermore, using the promoter base sequence and terminator sequence, the biodegradable plastic-degrading enzyme (PaE mass production system derived from P. antarctica) was successfully constructed, and the present invention was completed.
  • the novel protein is secreted and produced at a high concentration in the culture solution in the presence of xylose, the secretion amount of the heterologous protein into the culture solution can be controlled by using the secretory signal sequence. Furthermore, by culturing in the presence of xylose, it was confirmed that other yeasts of the genus Psudozyma also secreted a protein with the same molecular weight of about 33 kDa in the culture medium as in the case of P. antarctica. .
  • the present invention provides a novel promoter nucleotide sequence upstream of the gene PANT_8c00118 derived from P. 18antarctica that induces the expression of downstream genes in the presence of xylose, in order to strongly and strictly control the expression of heterologous protein genes in Pseudozyma spp.
  • An important feature is that it is used as a new promoter and enhances the secretion and productivity of heterologous proteins by placing or replacing the new signal sequence of PANT_8c00118 as a secretory signal sequence of the heterologous protein upstream of the target protein gene. And includes the following aspects.
  • a nucleic acid molecule comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 7.
  • An expression vector comprising the nucleic acid molecule according to any one of (1) to (3) above or a fragment thereof having promoter activity.
  • the expression vector according to (4) further comprising a sequence.
  • the expression vector according to (4) or (5) above further comprising a base sequence encoding a heterologous protein operably linked to the nucleic acid molecule.
  • the heterologous protein is a biodegradable plastic degrading enzyme PaE derived from yeast of the genus Pseudozyma or a biodegradable plastic degrading enzyme PCLE derived from the filamentous fungus NITE P-573.
  • (12) a step of culturing the host cell according to any one of (9) to (11) above under conditions that allow the expression of the heterologous protein, and a step of recovering the heterologous protein from the culture solution.
  • a method for producing a heterologous protein (13) The production method according to (12) above, wherein the host cell is cultured in a medium containing xylose (14) A protein encoded by the base sequence represented by SEQ ID NO: 4.
  • a biodegradable plastic degradation preparation comprising the protein according to (14).
  • heterologous proteins can be efficiently used with Pseudozyma yeast (for example, P. anatarctica) as a host.
  • Pseudozyma yeast for example, P. anatarctica
  • the remarkable effect is that it can be produced and accumulated in the medium.
  • the production amount was about 10 times higher than the production amount of the parent strain having an origin isolated from rice straw.
  • the target heterologous protein since the main component of the protein composition of the culture supernatant of the present invention is the target heterologous protein, it can be used directly as an enzyme solution by removing cells that are recombinants with a membrane filter or the like. .
  • M: marker, 1: reference strain P. antarctica JCM10317 strain, 2-10: isolated rice straw P. antarctica, 11: P. antarcticaT34 strain Results of SDS-PAGE and CBB staining of Pseudozyma yeast and Ustilago yeast culture with modified FMM using xylose as carbon source.
  • P. antarctica T34 strain This shows the PANT_8c00118 gene sequence of P. antarctica T34 strain and the base sequence including the promoter region and terminator region (SEQ ID NO: 5).
  • the base sequence in the frame is the base sequence of the structural gene of the PANT_8c00118 gene (SEQ ID NO: 4). Intron sequences are underlined and the start codon ATG and stop codon TGA are further framed.
  • the production of pPAX1-neo is shown.
  • the production of pPAX1-neo-PaCLE1 is shown.
  • Figure 2 shows halo formation by PBSA emulsion degradation.
  • the production of pPAXn-PCLE is shown.
  • Figure 2 shows halo formation by PBSA emulsion degradation.
  • the time-dependent changes in dry cell weight and enzyme activity of pPAXn-PCLE transformant (GB4 (0) -PCLE21 strain) of P. antarctica GB-4 (0) strain in 5L Jar fermenter are shown.
  • the base sequence in the frame is the base sequence of the structural gene of the xylose-derived protein. Intron sequences are underlined and the start codon ATG and stop codon TGA are further framed.
  • the present invention relates to a nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 1.
  • the base sequence shown in SEQ ID NO: 1 is a sequence existing upstream of a base sequence (SEQ ID NO: 4) encoding a protein having a molecular weight of about 33 kDa newly found in yeast of Pseudozyma antarctica.
  • the present invention further relates to a nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 1 and having promoter activity.
  • the base sequence is preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 and the base sequence shown in SEQ ID NO: 1. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
  • the base sequence has promoter activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added. More preferably, the nucleic acid molecule of the present invention consists of the base sequence shown in SEQ ID NO: 1.
  • the present invention relates to a nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 7.
  • the base sequence shown in SEQ ID NO: 7 is a sequence existing upstream of the base sequence (SEQ ID NO: 10) encoding a protein of about 33 kDa molecular weight newly found in the yeast GB4 (0) strain of Pseudozyma antarctica. is there.
  • the present invention further relates to a nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 7, and having promoter activity.
  • the base sequence is preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 and the base sequence shown in SEQ ID NO: 7. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
  • the base sequence has promoter activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added. More preferably, the nucleic acid molecule of the present invention consists of the base sequence shown in SEQ ID NO: 7.
  • the present invention further relates to an expression vector comprising the above nucleic acid molecule or a fragment thereof having promoter activity.
  • a fragment of the nucleic acid molecule of the present invention having promoter activity can be appropriately selected by a person skilled in the art by examining the promoter activity of the fragment according to a well-known method.
  • the length (base length) of the above fragment is not particularly limited as long as it has promoter activity, and is 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 bases or more. It is preferable. More preferably, the length of the fragment is 555, 560, 565, 570, 575, 580, 585, or 590 bases or more.
  • telomere sequences of 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 bases or more included in SEQ ID NO: 1 or SEQ ID NO: 7 are repeatedly linked.
  • Vectors suitable for gene expression are known in the art, and can be appropriately selected by those skilled in the art in consideration of combinations with host cells described below.
  • an expression vector for example, a plasmid pUXV1 (available from ATCC) or a chromosomal transfer vector can be appropriately modified as necessary.
  • the expression vector of the present invention is a terminator sequence of the PANT_8c00118 gene defined in SEQ ID NO: 2, or a sequence defined in SEQ ID NO: 8 (terminator sequence of a PANT_8c00118 homologous gene derived from the GB-4 (0) strain of the same Pseudozyma antarctica) ) May be included.
  • the expression vector of the present invention has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8.
  • % 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, It may contain a nucleotide sequence having 95%, 96%, 97%, 98%, 99% homology and having terminator activity. As long as the base sequence has terminator activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added.
  • the expression vector of the present invention may further comprise a base sequence encoding a heterologous protein operably linked to the nucleic acid molecule.
  • “operably linked” means that transcription of a heterologous protein is regulated under the influence of the promoter activity of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7 in an expression vector. It means that it is connected.
  • the heterologous protein can be appropriately selected by those skilled in the art as to the protein that needs to be expressed, and is not limited to the biodegradable plastic degrading enzyme shown in the Examples.
  • Preferable heterologous proteins include, for example, biodegradable plastic degrading enzyme PaE derived from yeast of the genus Pseudozyma or biodegradable plastic degrading enzyme PCLE derived from filamentous fungus NITE P-573.
  • the above base sequence may be a cDNA base sequence.
  • the expression vector of the present invention is adjacent to the upstream of the base sequence encoding the heterologous protein, and is shown in SEQ ID NO: 3 or SEQ ID NO: 9, or shown in SEQ ID NO: 3 or SEQ ID NO: 9.
  • SEQ ID NO: 3 is considered to correspond to the secretory signal sequence of the protein encoded from the start codon of the PANT_8c00118 gene (SEQ ID NO: 4) to 102 bp, and SEQ ID NO: 9 is the same Pseudozyma antarctica GB-4 (0 It is considered that it corresponds to the secretory signal sequence of the protein encoded from the start codon of the PANT_8c00118 homologous sequence (SEQ ID NO: 10) derived from the strain to 103 bp.
  • the target heterologous protein can be highly secreted and produced outside the microbial cell using a host cell transformed with the expression vector.
  • a promoter of a PANT_8c00118 gene, a heterologous protein cDNA base sequence, a terminator (eg, PANT_8c00118 gene terminator) gene base sequence is arranged, and a marker gene base sequence (eg, amino acid or It is possible to construct an expression cassette with a base synthesis gene and an antibiotic system gene).
  • the present invention further relates to a host cell transformed with the above expression vector.
  • the host cell can be appropriately selected by those skilled in the art in consideration of the combination with the expression vector to be used, and P. antarctica and its related species Pseudozyma genus yeast, such as P. antarctica, P. tsukubaensis, Examples include yeasts of the genus Pseudozyma such as P. rugulosa and P. aphidis, and Ustilaginales such as Ustilago genus such as Ustilago maydis.
  • yeasts belonging to the genus Pseudozyma are preferable, and Pseudozyma antarctica is particularly preferable.
  • the present invention further includes a step of culturing the above host cell containing a base sequence encoding a heterologous protein under conditions that allow expression of the heterologous protein, and a step of recovering the heterologous protein from the culture solution.
  • the present invention relates to a method for producing a heterologous protein.
  • the culture conditions can determine the composition of the medium and the culture temperature depending on the type of the vector and the host cell, and culture can be performed at 25 to 40 ° C., preferably 30 ° C.
  • the heterologous protein can be highly expressed by culturing host cells in a medium containing xylose.
  • Xylose can be added to the medium in an amount of 2 to 12% by mass, preferably 4 to 8% by mass.
  • the culture is carried out while keeping the aeration conditions at 0.5-3 vvm (LPM / L), preferably 1-2 vvm, and maintaining the dissolved oxygen concentration at 20-50% of the saturation.
  • LPM / L 0.5-3 vvm
  • an inexpensive carbon source instead of xylose, for example, sucrose, glycerol is sufficient to obtain 2 to 4% by mass in the medium, or an equivalent amount of microbial cells.
  • xylose with a final concentration of 10-20% by mass is continuously fed to induce enzyme production. Production is possible.
  • the recovery of the heterologous protein from the culture solution can be performed by any method.
  • ammonium sulfate may be added to the supernatant of the culture solution to obtain a precipitate, or the culture solution may be concentrated using a ultrafiltration method using Asahi Kasei Microza or the like.
  • the precipitate may be dialyzed with a 20 mM Tris-HCl buffer or the like using a dialysis tube, and then the coexisting protein may be removed by passing through a DEAE-sepharose column, and if necessary, SP-sepharose.
  • the enzyme may be purified through a column.
  • concentration and contaminant removal may be performed in one step using the property (affinity) that the heterologous protein binds to the substrate.
  • the present invention further relates to a protein encoded by the base sequence represented by SEQ ID NO: 4 and a biodegradable plastic degradation preparation containing the protein.
  • Biodegradable plastic degradation preparations can be prepared by those skilled in the art by well-known methods, and can also be mixed with well-known solvents, additives and the like. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by this.
  • antarctica strains owned by the present inventors secrete unknown proteins having a molecular weight of about 33 kDa in the presence of xylose in the same manner as the reference strain.
  • What was cultured in YM medium at 30 ° C. for 24 hours was used as a preculture solution.
  • a modified FMM was used to sterilize the carbon source (8% xylose) and other medium components separately and mix under sterilized conditions in order to avoid the formation of culture inhibitors such as browning and furfural. .
  • Each composition is shown below.
  • YM medium Yeast extract Malt extract Medium
  • Modified FMM Frazier minimum medium
  • NaNO 3 0.2%
  • a new gene sequence encoding this unknown protein was obtained.
  • DNA was extracted from the cells and physically fragmented, and then a Mate-pair library having an inserted fragment length of 3 to 4 kb was prepared.
  • the obtained Mate-pair library was mixed with microbeads for DNA addition, and the Mate-pair fragment was amplified by emulsion PCR, and then the microbeads were filled in a picotiter plate and aligned.
  • This picotiter plate was attached to a DNA sequencer (FLX Titanium), and the genomic DNA sequence was analyzed.
  • the obtained primary data was used to assemble a sequence to obtain a contig sequence, and the putative translation region on the genomic DNA sequence was comprehensively extracted by homology search.
  • the amino acid sequence was estimated from the gene sequence, and the molecular weight from the N-terminal amino acid sequence of the secreted protein was estimated to be 32,899.48 Da, which was consistent with the value of SDS-PAGE, confirming that the unknown protein was a protein encoded by PANT_8c00118 It was.
  • the protein from the start codon is determined based on the N-terminal amino acid sequence of this protein purified from the culture solution. It was speculated that there was a strong secretion signal in 102 bp immediately before the gene sequence encoding the N-terminal amino acid sequence.
  • FIG. 5 shows that this plasmid incorporates the cDNA gene PaCLE1 encoding the biodegradable plastic-degrading enzyme PaE derived from P. antarctica JCM10317.
  • the constructed plasmid pPAX1-neo-PaCLE1 was introduced into the P. antarctica T34 strain according to the following method.
  • the recovered cells were suspended in 10 ml of ice-cold 1M sorbitol, and centrifuged at 4 ° C. and 3000 ⁇ g for 5 minutes to recover the cells.
  • the collected cells were suspended in 1 ml of ice-cold 1M sorbitol.
  • the above operation was performed on ice.
  • 50 ⁇ l of the cell suspension and 50 ⁇ l of plasmid DNA (about 5 to 10 ⁇ g) were added to an electroporation cuvette (BIO-RAD) with a gap of 0.2 cm and incubated on ice for 5 minutes.
  • a cuvette was set in E. coli pulser (BIO-RAD), and electroporation was performed under conditions of 1.5 kV, 25 kF, and 400 ⁇ .
  • ice-cold YMS (YM + 1M sorbitol) was immediately added, mixed by pipetting, transferred to a 15 ml polyurethane tube, and incubated at 30 ° C. for 2 hours. Apply to a YMS plate containing antibiotic G418 (YMS + 0.05% G418, 2% agar) and incubate at 30 ° C until colonies form. The resulting transformant colonies contain the new antibiotic G418. Planted on YMS plate.
  • Transformants grown on the YMS medium containing antibiotics were added to FMM plates containing PBSA emulsion (0.1% yeast extract, 0.2% NaNO 3 , 0.02% KH 2 PO 4 , 0.02% MgSO 4 .7H 2 O, 2 % Xylose, 1% PBSA emulsion (EM-301, Showa Denko KK, 2% agar), and statically cultured at 30 ° C.
  • the T34 strain used as the parent strain does not produce biodegradable plastic-degrading enzyme PaE in the presence of xylose, but the transformant transforms the cloudy PBSA emulsion in the medium due to the activity of PaE induced by xylose.
  • a transparent halo is formed ( Figure 6). Such a transformant was selected.
  • transformants incorporating the previously selected pPAX1-neo-PaCLE1 were transformed into 1% yeast extract solution with 6% of various sugar sources (glucose, When cultured in media supplemented with galactose, xylose, arabinose, sucrose, and lactose, respectively, it was revealed that PBSA emulsion degradation activity was strongly induced only when xylose was used (FIG. 7).
  • the enzyme activity was measured as follows.
  • rugulosa JCM10323 which are yeasts belonging to the genus Pseudozyma, in the same manner as above, all strains of FMM containing PBSA emulsion
  • the emulsion was dissolved in the vicinity of the bacteria to obtain a transformant that formed halo (see FIG. 8, transformant of P. rugulosa JCM10323 strain), and 6% in 1% yeast extract
  • PBSA emulsion degradation activity was strongly induced in the culture filtrate by culturing in a liquid medium containing xylose (see FIG. 9, transformants of P. tsukubaensis JCM10324 and P. aphidis JCM10318).
  • xylose fed-batch culture was performed using GB4 (0) -PaE14 strain.
  • 3 L of medium with the following composition with low initial xylose concentration was added to 5 L Jar fermenter, inoculated with 30 ml of preculture, and cultured at 30 ° C. with stirring speed of 500 rpm and aeration rate of 8 LPM .
  • a 50-fold diluted disinfectant Shin-Etsu Silicone KM-72F, Shin-Etsu Chemical Co., Ltd.
  • Enzyme activity increased with the addition of fed-batch medium, and after 72 hours, 56.9 U / mL of enzyme was produced in the culture supernatant, but the subsequent enzyme production rate slowed down to 104 hours. Later, the maximum activity was 64.1 U / mL (see FIG. 10A).
  • the cause was thought to be a lack of nutrient sources other than xylose in the medium.
  • the protein production rate at the initial stage of the culture was low, and the protein production rate increased rapidly after 24 hours of culturing when the cells grew to some extent. It was decided to perform high-density fed-batch culture in a state where the protein production rate was kept high by performing only cell growth using the source and feeding xylose from 24 hours onward.
  • the medium composition for high-density culture and the feed medium composition are shown below. Other culture conditions were the same as described above.
  • Medium composition for high density culture in Jar fermenter Sucrose 3% NaNO 3 0.2% MgSO 4 ⁇ 7H 2 O 0.1% KH 2 PO 4 0.1% Yeast extract 0.5% (NH 4 ) 2 SO 4 1.0%
  • Fed-batch medium composition for high-density culture Xylose 50% YNB w / o AA & AS 0.085% Yeast extract 0.2% The dry cell weight grew to 22.6 g / L in 24 hours from the start of the culture, and then maintained a high protein production rate from the start of the culture to 104 hours by the addition of xylose.
  • a very high concentration of PaE of mL (about 3 g / L) was obtained (see FIG. 10B). This value was about 10 times higher than that of the parent strain.
  • FIG. 11 shows pPAXn-PCLE in which the developed expression vector UARS was deleted by restriction enzyme SspI treatment and a cDNA gene sequence encoding PCLE (GenBank Accession No. AB823703) was incorporated. After pPAXn-PCLE was linearized by treatment with the restriction enzyme EcoRI, a transformant, T34-PCLE1 strain, incorporated into the P. antarctica T34 strain was prepared.
  • T34 and T34-PCLE1 strains were inoculated on the FMM plate containing the PBSA emulsion described above, the T34-PCLE1 strain was decomposed by the production of PCLE, and the formation of halo was confirmed as shown in FIG. .
  • GB-4 (0) -PCLE3 strain in which pPAXn-PCLE was incorporated into the GB-4 (0) strain was prepared in the same manner.
  • the PBSA emulsion degrading activity of the parent strain GB-4 (0) was 1.8 U / ml due to the activity of the parent strain derived PaE after 96 hours of shaking culture.
  • GB-4 (0) -PCLE3 strain showed 2.6 U / ml, and the value was remarkably increased by the production of PCLE.
  • the enzyme PCLE of the filamentous fungus B47-9 strain has a dramatic increase in PBSA emulsion degradation activity by the addition of CaCl 2 (Suzuki K., et al., Appl. Microbiol. Biotechnol. DOI10.1007 / s00253). -013-5454-0), PBSA emulsion degradation activity with CaCl 2 added to 1 mM was measured.
  • the parent strain GB-4 (0) was 3.5 U / ml
  • GB- The strain of 4 (0) -PCLE3 was 5.8 U / ml, and the effect of increasing PCLE activity by adding CaCl 2 was confirmed.
  • XynF1 5'-GAAGGCTGAAGCTTTGGCTCTGACAT-3 '
  • XynF2 5'-CATGCTTGAAGCTCCAAGAAGATATAA-3
  • XynF3 5'-CACTCGCAGCTGCCTTCGTGGGTGCAG-3
  • XynF4 5'-GAAGGCAGTCTGCTCGGCCGCTCCCGA-3 '
  • XynR1 5'-TGTGGTGTTTGTTTGGCGTTTTTGCTT-3 '
  • XynR2 5'-ATCCCACGCGTACACCTTGCCCTTGTA-3 '
  • XynR3 5'-CCCACGCAGTTGGACTGGGCCAGGCAG-3 '
  • XynR4 5'-CGAGCGCGATTTTCTCCGAGTCTAAA-3 '
  • the gene sequence of 1954 residues (SEQ ID NO: 11) shown in FIG. 14 was obtained from the genomic DNA of GB-4 (0) by direct PCR.
  • This gene sequence shows 88% homology with the PANT_8c00118 gene promoter, structural gene, and terminator sequence (FIG. 3, SEQ ID NO: 5) of the T34 strain as a whole, and the structural gene sequence encoding the protein (SEQ ID NO: 10) Showed 94% homology with the sequence of the T34 strain (SEQ ID NO: 4).
  • the promoter region (SEQ ID NO: 7) shows 83% homology with that of the T34 strain (SEQ ID NO: 1)
  • the terminator region (SEQ ID NO: 8) has that of the T34 strain (SEQ ID NO: 2).
  • the signal sequence (SEQ ID NO: 9) showed 93% homology with that of the T34 strain (SEQ ID NO: 3).

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Abstract

L'invention concerne un procédé qui comprend le développement d'un promoteur à haute expression de Pseudozyma antarctica, la construction d'un système de production de protéine hétérologue à l'aide du promoteur à expression élevée obtenu, puis la production efficace d'une enzyme à l'aide d'une souche appartenant à l'ordre Ustilaginales comprenant une levure du genre Pseudozyma. Le procédé de production de protéine hétérologue selon la présente invention comprend : une étape de culture de cellules hôtes, qui ont été transformées par un vecteur d'expression qui contient une séquence de base représentée par SEQ ID n°1 ou SEQ ID n°7 et une séquence de base, ladite séquence de base codant pour une protéine hétérologue, liée de façon fonctionnelle à celle-ci, dans des conditions telles qu'elles permettent l'expression de la protéine hétérologue ; et une étape de récolte de la protéine hétérologue à partir d'un milieu de culture liquide.
PCT/JP2014/050233 2013-01-09 2014-01-09 Procédé pour la production hautement efficace d'une protéine hétérologue WO2014109360A1 (fr)

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JP2018157814A (ja) * 2017-03-21 2018-10-11 国立研究開発法人農業・食品産業技術総合研究機構 シュードザイマ・アンタクティカの新規菌株
JP2022049902A (ja) * 2020-09-17 2022-03-30 国立研究開発法人農業・食品産業技術総合研究機構 キシロース誘導性プロモーター及びその使用
WO2023140361A1 (fr) * 2022-01-21 2023-07-27 国立研究開発法人農業・食品産業技術総合研究機構 Facteur de transcription et son utilisation
WO2023176835A1 (fr) * 2022-03-16 2023-09-21 国立研究開発法人農業・食品産業技術総合研究機構 Promoteur inductible par le xylose et son utilisation

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017204288A1 (fr) * 2016-05-25 2017-11-30 国立研究開発法人農業・食品産業技術総合研究機構 Composition pour améliorer la résistance aux maladies de plantes ou lutter contre une maladie de plante, et son procédé d'utilisation
JPWO2017204288A1 (ja) * 2016-05-25 2019-04-18 国立研究開発法人農業・食品産業技術総合研究機構 植物の病害抵抗性増強用又は植物病害防除用組成物及びそれらの使用方法
JP6997456B2 (ja) 2016-05-25 2022-01-17 国立研究開発法人農業・食品産業技術総合研究機構 植物の病害抵抗性増強用又は植物病害防除用組成物及びそれらの使用方法
JP2018157814A (ja) * 2017-03-21 2018-10-11 国立研究開発法人農業・食品産業技術総合研究機構 シュードザイマ・アンタクティカの新規菌株
JP2022126864A (ja) * 2017-03-21 2022-08-30 国立研究開発法人農業・食品産業技術総合研究機構 シュードザイマ・アンタクティカの新規菌株
JP7181542B2 (ja) 2017-03-21 2022-12-01 国立研究開発法人農業・食品産業技術総合研究機構 シュードザイマ・アンタクティカの新規菌株
JP2022049902A (ja) * 2020-09-17 2022-03-30 国立研究開発法人農業・食品産業技術総合研究機構 キシロース誘導性プロモーター及びその使用
JP7299623B2 (ja) 2020-09-17 2023-06-28 国立研究開発法人農業・食品産業技術総合研究機構 キシロース誘導性プロモーター及びその使用
WO2023140361A1 (fr) * 2022-01-21 2023-07-27 国立研究開発法人農業・食品産業技術総合研究機構 Facteur de transcription et son utilisation
WO2023176835A1 (fr) * 2022-03-16 2023-09-21 国立研究開発法人農業・食品産業技術総合研究機構 Promoteur inductible par le xylose et son utilisation

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