WO2003016525A9 - Procede de production d'alcool a partir d'amidon - Google Patents

Procede de production d'alcool a partir d'amidon

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Publication number
WO2003016525A9
WO2003016525A9 PCT/JP2002/008234 JP0208234W WO03016525A9 WO 2003016525 A9 WO2003016525 A9 WO 2003016525A9 JP 0208234 W JP0208234 W JP 0208234W WO 03016525 A9 WO03016525 A9 WO 03016525A9
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WO
WIPO (PCT)
Prior art keywords
yeast
amylase
cell surface
starch
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2002/008234
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English (en)
Japanese (ja)
Other versions
WO2003016525A1 (fr
Inventor
Hideki Fukuda
Akihiko Kondo
Atsuo Tanaka
Mitsuyoshi Ueda
Eiichi Satoh
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Kansai Chemical Engineering Co Ltd
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Kansai Chemical Engineering Co Ltd
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Filing date
Publication date
Priority claimed from PCT/JP2002/001980 external-priority patent/WO2003016524A1/fr
Application filed by Kansai Chemical Engineering Co Ltd filed Critical Kansai Chemical Engineering Co Ltd
Priority to JP2003521834A priority Critical patent/JP4189317B2/ja
Publication of WO2003016525A1 publication Critical patent/WO2003016525A1/fr
Publication of WO2003016525A9 publication Critical patent/WO2003016525A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a method for producing ethanol from starch. More specifically, the present invention relates to a method for producing ethanol using yeast which presents glycoamylase on the cell surface and secretes ⁇ -amylase. Alternatively, the present invention relates to a method for producing ethanol using yeast that displays dalcoamylase and ⁇ -amylase as a fusion protein on the cell surface. More specifically, the present invention relates to a method for producing ethanol from uncooked starch using the yeast as described above. Background art
  • biomass as a new energy resource has attracted attention in recent years.
  • Cellulose and starchy substances of plant origin are the most abundant and available biomass resources.
  • ethanol produced from starch resources is attracting attention as a renewable environmentally-friendly energy resource, and the demand for it is expected to increase in the future.
  • Ethanol production from starch by the current fermentation method is carried out in two stages: starch is steamed, starch is saccharified by treatment with koji mold that secretes amylase, and fermentation is performed by fermentation. . This is because yeast does not have amylases and therefore cannot degrade and saccharify starch.
  • an object of the present invention is to construct a yeast capable of producing ethanol directly and more efficiently from starch, and to provide a method for producing ethanol using the yeast.
  • the present inventors transformed yeast to display dalcoamylase on the cell surface and to secrete or display ⁇ -amylase on the cell surface. As a result, it was found that ethanol productivity was significantly improved, and the present invention was completed. Accordingly, the present invention provides a yeast which displays dalcoamylase on the cell surface and secretes ⁇ -amylase.
  • the dalcoamylase is presented via a GPI anchor.
  • the dalcoamylase is presented as a fusion protein with a sugar chain binding protein domain that does not have a GP I anchor attachment recognition signal sequence.
  • the present invention also provides a darcoamylase and ⁇ -amylase fusion protein
  • a yeast which is displayed on a cell surface as a fusion protein with a sugar chain binding protein domain having no G ⁇ I anchor attachment recognition signal sequence, wherein the dalcoamylase is fused to one end of the domain, and the other end is the yeast.
  • Hi-amylase Provide yeast that has been fused.
  • the sugar chain binding protein domain is a portion containing at least an aggregation functional domain of a GP I anchor protein.
  • the GPI anchor protein is an aggregated protein.
  • the aggregated protein is a protein selected from the group consisting of FL01, FL02, FL04, FL05, FLO9, FLO10, and FLO11.
  • the ⁇ -amylase is from Streptococcus bovis 148.
  • the present invention also provides a method for producing alcohol, comprising a step of culturing the yeast in a medium containing starch.
  • the starch is uncooked.
  • FIG. 1 is a schematic diagram showing the construction of a plasmid pAAl2 that presents ⁇ -amylase on the cell surface by a GPI anchor.
  • FIG. 2 is a schematic diagram showing the construction of plasmid pSAA11 that secretes ⁇ -amylase.
  • FIG. 3 is a schematic diagram showing the construction of plasmid pSGAl1, which secretes dalcoamylase.
  • FIG. 4 is a schematic diagram of the plasmid BAA1.
  • FIG. 5 is a schematic diagram of the plasmid pSBAA2.
  • FIG. 6 is a graph showing changes over time in starch concentration and cell density when various recombinant yeast cells are cultured under aerobic conditions.
  • Figure 7 shows the results of alcohol fermentation using uncooked corn starch medium. It is a graph which shows a time-dependent change of the starch density
  • FIG. 8 is a graph showing the time-dependent changes in the starch concentration and the ethanol concentration in the medium when various concentrations of non-steamed corn starch are used.
  • FIG. 9 is a graph showing the time-dependent changes in the starch concentration and the ethanol concentration in the medium when various concentrations of yeast YF207 / [pGA11, pSBAA2] were used.
  • FIG. 10 is a plate photograph showing the results of a plate assay on glucoamylase activity.
  • FIG. 11 is a photograph of a plate showing the results of plate assay for ⁇ -amylase activity.
  • dalcoamylase refers to an exo-type hydrolase that separates a glucose unit from the non-reducing end of starch.
  • the origin is not limited as long as it has such an activity.
  • darcoamylase derived from molds such as Rhizopus and Aspergillus is used.
  • darcoamylase derived from Rhizopu s oryzae is preferably used.
  • ⁇ -amylase refers to an endo-type enzyme that hydrolyzes ⁇ 1,4-darcoside bonds of starch.
  • the source of the activity is not limited as long as it has this activity.
  • ⁇ -amylase derived from animals (such as saliva and kidney), plants (such as malt), and microorganisms is used.
  • alpha-amylase force derived from a microorganism s preferred include those such as from Streptococcus bovis.
  • ⁇ -amylase derived from Streptococcus bovis is particularly preferred when uncooked starch is used as a carbon source.
  • the first yeast of the present invention is yeast transformed to present dalcoamylase on the cell surface and secrete ⁇ -amylase.
  • Such a yeast can be obtained by introducing into a yeast D ⁇ ⁇ ⁇ ⁇ recombined to display glucoamylase on the cell surface and D ⁇ ⁇ ⁇ ⁇ recombined to secrete ⁇ -amylase. , can get.
  • the second yeast of the present invention is a yeast transformed to display dalcoamylase and ⁇ -amylase as a fusion protein with a sugar chain binding protein domain on a cell surface.
  • Such yeasts can be obtained by introducing into the yeast a recombinant DNA that displays the darcoamylase, ⁇ -amylase, and carbohydrate-binding protein domains as a single fusion protein on the cell surface.
  • the method of presenting the enzyme on the cell surface includes: (a) a method of presenting the enzyme to the cell surface via a GPI anchor of a cell surface-localized protein; and (b) a method of displaying the sugar on the cell surface-localized protein.
  • any of the methods (a) and (b) may be used.
  • Cell surface localization proteins that can be used include yeast sex aggregation proteins ⁇ - or a-agglutune, FLO proteins (eg, FL01, FLO2, FL04, FL05, FL09, FLO10, and FLOl 1), alkaline phosphatase And the like.
  • Genes encoding proteins localized on the cell surface by the GPI anchor include, in order from the N-terminus, a secretory signal sequence, a cell surface localized protein (sugar chain binding protein domain), and a GPI anchor attachment recognition signal sequence. Each has an encoding gene.
  • the cell surface localization protein (sugar chain binding protein) expressed from this gene in the cell is guided out of the cell membrane by a secretory signal, and the GPI anchor attachment recognition signal sequence is selectively cleaved. It is fixed to the cell membrane by binding to the GPI anchor of the cell membrane via the terminal part. Thereafter, the root of the GPI anchor is cut off by PI-PLC, incorporated into the cell wall, fixed to the cell surface, and presented to the cell surface.
  • the GPI anchor refers to a glycosylphosphatidylinositol (GPI) ethanolamine phosphate-6 mannose ⁇ 1-2 mannose 1-6 mannose a 1-4 darcosamiso a 16 inositol phospholipid basic structure
  • PI-PLC refers to phosphatidylinositol-dependent phospholipase C.
  • the GPI anchor attachment recognition signal sequence is a sequence that is recognized when the GPI anchor binds to a cell surface localization protein, and is usually located at or near the C-terminus of a cell surface localization protein.
  • the GPI anchor attachment signal sequence for example, the sequence of the C-terminal part of yeast agglutin is preferably used.
  • a GPI anchor attachment recognition signal sequence is included on the C-terminal side of the sequence of C-terminal 320 amino acids from the C-terminal of the ⁇ -agglutene. DNA sequences encoding are particularly useful.
  • the structural gene encoding a cell surface localized protein the cell surface localized protein Recombinant DNA that displays the target enzyme on the cell surface via a GPI anchor by replacing the sequence of all or part of the structural gene encoding the target enzyme with the sequence of the structural gene of the target enzyme Is obtained.
  • a glucoamylase gene is used as the structural gene of the enzyme of interest, a recombinant DNA that presents dalcoamylase on the cell surface via a GPI anchor can be obtained.
  • the sugar chain-binding protein domain has a plurality of sugar chains, and the sugar chains interact with or are entangled with the sugar chains in the cell wall. It is possible to stay.
  • a sugar chain binding site of lectin, lectin-like protein and the like can be mentioned.
  • a typical example is the aggregation functional domain of a GPI anchor protein.
  • the aggregation functional domain of the GPI anchor protein refers to a domain that is located on the N-terminal side of the GPI anchoring domain, has a plurality of sugar chains, and is considered to be involved in aggregation.
  • the enzyme is displayed on the cell surface by binding the cell surface localized protein (aggregation function domain) with the target enzyme.
  • the enzyme is bound to the N-terminal side of the cell surface localized protein (aggregation functional domain)
  • the enzyme is bound to the C-terminal side
  • the N-terminal side is bound to both the C-terminal side and the C-terminal side.
  • DNA encoding a secretory signal sequence—structural gene of the enzyme of interest Structural gene encoding a single cell surface localization protein (aggregation function domain)
  • DNA encoding a secretory signal sequence a structural gene encoding a cell surface localization protein (aggregation function domain) —a structural gene of an enzyme of interest;
  • a recombinant DNA that displays dalcoamylase on the cell surface using the sugar chain binding protein domain can be obtained.
  • a secretory signal sequence of a protein localized on the cell surface may be used, or another secretory signal sequence capable of directing the expressed enzyme to the outside of the cell may be used. Is also good.
  • a secretory signal sequence for dalcoamylase, a secretory signal sequence for yeast ⁇ - or a-agglutin, and a secretory signal sequence for lipase are preferably used. If the enzyme activity is not affected, part or all of the secretory signal sequence and prosequence may remain at the N-terminus after cell surface display.
  • a recombinant DN DN in which a structural gene of a target enzyme such as dalcoamylase or ⁇ -amylase is linked to DNA encoding the above-mentioned secretory signal sequence may be introduced into yeast.
  • DNAs containing the above-described various sequences can be performed by those skilled in the art using conventional techniques. For example, binding of the secretory signal sequence to the structural gene of dalcoamylase or human amylase is performed using site-directed mutagenesis. I can. By using this method, accurate secretory signal sequence cleavage and expression of active dalcoamylase or monoamylase are possible.
  • the above sequence of interest is preferably incorporated into a vector.
  • a shuttle vector with Escherichia coli is preferable.
  • it has a replication origin (Or i) of 2 m plasmid of yeast and a replication origin of ColEl, and further has a yeast replication origin.
  • it has a selection marker (eg, drug resistance gene, TRP, LEU2, etc.) and an E. coli selection marker (eg, drug resistance gene).
  • the dalcoamylase or ⁇ -amylase structural gene it is desirable to include so-called regulatory sequences such as an operator, a promoter, a terminator, and an enhancer that regulate the expression of this gene.
  • regulatory sequences such as an operator, a promoter, a terminator, and an enhancer that regulate the expression of this gene.
  • the plasmid pYGA2270 or pYE 22m containing the GAPDH (glyceraldehyde 3, monophosphate dehydrogenase) promoter and the GAPDH terminator, or the UPR-ICL (isoquenate lyase upstream region) sequence
  • a plasmid pWI3 containing a Term-ICL (terminator region of isocitrate lyase) sequence.
  • a DNA encoding a secretory signal sequence between the sequence of the GAP DH promoter and the GAP DH terminator of the plasmid pYG A2270 or p YE 22 m By inserting a sequence that combines the sequence of the darco amylase or ⁇ -amylase structural gene and the sequence encoding 320 amino acids from the C-terminus of ⁇ -agglutinin, a binder for introduction into yeast can be produced. Is done.
  • Vectors are available in multicopy and chromosomal integration types. Which type of vector is to be incorporated into which gene may be appropriately determined by those skilled in the art.
  • the enzyme displayed on the cell surface and the secreted enzyme may be incorporated in the same vector, PT / JP02 / 08234
  • Each may be integrated into a different vector.
  • any yeast can be used as long as it can utilize sugar to have alcohol fermentation ability.
  • Non-aggregating and flocculating yeasts are used.
  • Cohesive yeasts are preferred because they can be separated easily after the reaction, or because they can be fixed easily and can be subjected to a continuous reaction.
  • the non-aggregating yeast is not particularly limited, and examples thereof include Saccharomyces ce revisiae MT8-1.
  • yeast of flocculation examples include Saccharomyces diastaticus ATCC60715 and ATCC60712, Saccharomyces cerevisiae IF01953, CG1945 and HF7C. Further, a new flocculant yeast may be constructed. For example, as shown in “Preparation of Experimental Materials” in Examples below, flocculent yeast was produced according to the method of MD Rose et al. (Methods in Yeast Genetics, 1990, Cold Spring Harbor Laboratory Press, Cold Spring Haroor, NY). From the diploid obtained by conjugation of ATCCS0712 with the non-aggregating yeast W303-IB, it is possible to obtain an aggregating yeast YF207 and a yeast having properties equivalent thereto.
  • the flocculent yeast strain YF207 obtained by the present inventors has excellent plasmid retention stability, and has very high fermentation ability. Therefore, when the flocculent yeast strain YF207 which is dal coamylase displayed on the cell surface and is recombinantly secreted to secrete ⁇ -amylase is used, the productivity of ethanol is extremely high.
  • a yeast which presents the glucoamylase of the present invention to the cell surface and secretes ⁇ -amylase, or a yeast which presents dalcoamylase and ⁇ -amylase to the cell surface as a fusion protein (hereinafter referred to as the yeast of the present invention and ) Can be obtained by simultaneously or separately introducing recombinant DNA (vector) having DNAs encoding the above enzymes into yeast.
  • Methods for introducing DN ⁇ include transformation, transduction, transfection, co-transfection, and electoral poration. Examples include a transformation method using lithium acetate and a protoplast method.
  • Yeast into which the recombinant DNA (vector) has been introduced is selected with a selectable marker (eg, TRP, URA).
  • the presence of dalcoamylase or ⁇ -amylase on the cell surface can be confirmed after washing the cells, for example, by an immunoantibody method using an anti-glucoamylase antibody or an anti-amylase antibody and a FITC-labeled antibody. .
  • the secretion of ⁇ -amylase can also be confirmed in a culture solution from which cells have been removed, for example, by an immunoantibody method using an anti-amylase antibody.
  • the yeast of the present invention may be immobilized on a carrier. When fixed, it is convenient for use in repeated batch or continuous fermentations.
  • a method usually used for yeast by those skilled in the art is applied.
  • the immobilized yeast can be used as a so-called bioreactor by being attached to a carrier and filled in a column that is cultured in a suspended state. Even if the fermentation is repeated continuously or in batches (batch), the activity of the yeast decreases or the dead yeast is eliminated, so that the yeast activity does not decrease and the yeast is effective. Can be used for
  • the yeast of the present invention is first cultured under aerobic conditions to increase its number.
  • the medium may be a selective medium or a non-selective medium.
  • This yeast can be grown using starch as a carbon source, and the concentration of starch in the culture medium during culturing is preferably about 1 to about 10 g / l, more preferably about 2 to about 6 g when soluble starch is used. g / l, most preferably about 4 g / l.
  • the concentration of starch in the medium is about 1 to about 50 g / l, preferably about 2 to about 40 g / 1, and more preferably about 10 to about 20 g / l.
  • the pH of the culture medium during the culturing is preferably about 4.0 to about 6.0, most preferably about 5.0. In medium during aerobic culture PT / JP02 / 08234
  • the dissolved oxygen concentration of 12 is preferably about 0.5 to about 6 ppm, more preferably about 1 to about 4 ppm, and most preferably about 2.0 ppm.
  • the temperature during the culturing is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C.
  • the culturing is preferably performed until the cell concentration reaches 10 g / l or more.
  • the yeast of this effort is fermented under anaerobic conditions to produce ethanol.
  • Examples of the form of this fermentation step include a batch (batch) step, a fed-batch batch step, a repeated batch step, a continuous step, and the like, and any of these may be used.
  • the batch fermentation process is a closed fermentation method performed by inoculating yeast into a medium previously placed in a fermenter.
  • fermentation is performed while supplying a nutrient medium to the batch process, but the target product is not extracted until a certain time.
  • the repeated batch process is a process in which the above batch process is repeated. Specifically, after the first batch process, the operation of separating the culture medium and yeast, extracting the culture medium, and then adding a fresh culture medium to perform the fermentation step is repeatedly performed.
  • the continuous fermentation process is a process in which fresh medium is continuously supplied to the fermenter while simultaneously extracting the medium containing the product (ie, ethanol) from the fermenter.
  • the starch concentration in the medium is preferably about 40 to about 150 g / l.
  • the starch concentration is more preferably from about 50 to about 120 g / l, most preferably about 60 g / l.
  • the added starch concentration is preferably maintained at about 40 to about 300 g / l, more preferably about 60 to about 250 g / l, most preferably about 200 g / l.
  • the starch concentration in the medium is about 50 to about 500 g / l, preferably about 50 to about 400 g / l, and more preferably about 150 to about 250 g / l.
  • the pH of the medium during fermentation is preferably from about 4.0 to about 6.0, most preferably about 5.0. ⁇
  • the dissolved oxygen concentration in the medium during aerial fermentation varies depending on the yeast used as the host, Preferably it is less than about 1.0 ppm, more preferably less than about 0.1 ppm, most preferably less than about 0.05 ⁇ .
  • the temperature during fermentation is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C.
  • the initial cell concentration in the medium during anaerobic fermentation (charge concentration), type of yeast, the force varies by as starch concentration of culture ground S, preferably early 0D 6. . Is from 10 to 300, more preferably from 30 to: L00.
  • the medium containing ethanol is withdrawn from the fermenter, and ethanol is isolated by a separation step commonly used by those skilled in the art such as a centrifugal separator and a distillation operation.
  • Saccharomyces diastaticus ATCC60712 (MATa leu2-3, 1 12 his2 lys2 stal FL08), which is an aggregating yeast, and W303-IB ( ⁇ ⁇ ura3-52 trpl A 2 leu2—3, 112 his3— 11, a non-aggregating yeast ade2—1 canl-100) and according to the method of MD Rose et al., supra, a new aggregating strain of tributofan auxotrophy, YF207 (MATa ura3-52 trpl ⁇ 2 his ade2-1 canl-). 100 stal FL08).
  • yeast Saccharomyces cerevisiae MT 8-1 (MATa ade his3 leu2 trpl ura3) (Tajima et al., Yeast, 1: 67-77, 1985) Used as yeast.
  • the multicopy plasmid pGAl1 which presents the dalcoamylase from Rhizopus oryzae on the cell surface, is described by the inventor Tanaka Ope Ueda et al. (Appi. And Environmental Microbiology (1997) 63: 1362-1366). The one described in was used.
  • Plasmid p AA12 which presents ⁇ -amylase from Bacillus stearothermophilus on the cell surface, was prepared using plasmid ⁇ I ⁇ 11 as a starting material.
  • Figure 1 shows a schematic diagram of the construction. Plasmid ⁇ ⁇ ⁇ 11 was treated with Xho I to separate it into a long fragment and a short fragment.
  • a plasmid obtained by self-ligating the long fragment was cut with NotI and KpnI and blunt-ended to obtain a fragment of about 860 bp.
  • This fragment has a GAP DH promoter sequence and a gene sequence encoding 320 amino acid residues from the 3 ′ side of ⁇ -agglutin.
  • the multicopy plasmid ⁇ 34 (+3) was digested with ⁇ VuII and BamHI, blunted, and the above fragment of about 860 bp was inserted into the plasmid to obtain plasmid pUGP1. I got 2.
  • a short fragment obtained by treating plasmid p I ⁇ 11 with Xho I was used to obtain a secretion signal sequence of about 2,000 b of the yeast a-functional enzyme.
  • ⁇ -amylase derived from Bacillus ste arothermophilus Encoding the mature protein sequence I have. This fragment was introduced into the XhoI site of the plasmid pUGP12 to obtain a plasmid pAAl2 used for displaying ⁇ -amylase on the cell surface.
  • Plasmid pSAAl1 which secretes ct-amylase from Bacillus stearothermophilus, is described in the literature of the inventor Tanaka Ope Ueda et al. (Murai et al., Appl. Microbiol. Biotechnol. (1999) 51: 65-70).
  • the ⁇ -amylase gene was isolated from the chromosome-integrated plasmid pIAAA1 of the above, and this was integrated into a multicopy plasmid: UGP 3 (Takahashi et al., Appl. Microbiol. Biotechnol. (2 001) 55: 454-462).
  • Figure 2 shows a schematic diagram of the construction.
  • Plasmid pSGA11 secreting darcoamylase from Rhizopus oryzae was prepared by the method shown in FIG. Using pGAl1 as a template and a primer, 5,-ATCGGGATCCATGCMCTGTTC TTTGCCATTGAAAGTT-3 '(sequence number 3) and 5,-ATCGGTCGACTTMGCGGCAGGTGCACCAGCCTTAGCGTA-3, (sequence number PCR amplification was carried out using No. 4), followed by digestion with restriction enzymes BamHI and Sa1I to obtain a BamHI-Sa1I fragment of about 1800 bp. This fragment encodes the secretory signal sequence of dalcoamylase and the mature protein sequence of dalcoamylase.
  • the multicopy plasmid p UGP3 (Takahashi et al., Supra) was digested with restriction enzymes BamHI and Sa1I, and the above-mentioned BamHI-Sa1I fragment of about 1800 bp was added thereto. Ligation produced the plasmid pSGAl1 used to secrete glycoamylase.
  • Plasmid pBAAl which presents ⁇ -amylase derived from Streptococcus bovis 148 on the cell surface was prepared using plasmid pAA12 obtained in the above [a-2] as a starting material.
  • Figure 4 shows a schematic diagram of the plasmid pBAAl.
  • the plasmid pAAl2 was treated with XhoI and separated into a long fragment (6.9 kb) and a short fragment (1.7 kb).
  • a plasmid obtained by self-ligating the long fragment was cut with NotI and KpnI and blunt-ended to obtain a fragment of about 860 bp.
  • This fragment has a GAPDH promoter sequence and a gene sequence encoding 320 amino acid residues from the 3 ′ side of ⁇ -agglutinin.
  • the multicopy plasmid ⁇ 34 (+3) was digested with ⁇ VuII and BamHI, blunted, and the above fragment of about 860 bp was incorporated to obtain plasmid pUGP12. Was.
  • ATTTGCCATTGAAAGT-3 (SEQ ID NO: 5) and 5, -CTGCCCATGGGGTTTTAGCCCATCTT TATTATAGTTTCC-3 '(SEQ ID NO: 6)
  • a 2.2 kb DNA fragment containing the ⁇ -amylase gene derived from Streptococcus bovis 148 was obtained. This fragment was introduced into the XhoI site of the plasmid pUGP12 to obtain BAAl, a plasmid used to display ⁇ -amylase derived from Streptococcus bovis on the cell surface.
  • FIG. 5 shows a schematic diagram of the plasmid pSBAA2 that secretes a-amylase derived from Streptococcus bovis 148.
  • Plasmid p CAS 1 Using amyA as a template, primers 5'-AATAGAGCTCATGCAACTGTTCAATTTGC CATTGAAAGT-3, (SEQ ID NO: 7) and 5'-TGGCGGTACCTTATTTTAGCCCATCTTTATTA TAGTTTC-3 '(SEQ ID NO: 8) Amplified by CR and digested with restriction enzymes SacI and KpnI, a 2.2 kb DNA fragment encoding the secretory signal sequence of dalcoamylase and the mature protein sequence of dalcoamylase was obtained.
  • the multicopy-type plasmid pUGP3 (Takaha shi et al., Supra) was digested with restriction enzymes SacI and KpnI, and the above SacI- n ⁇ nI fragment was ligated therewith, thereby obtaining Streptococcus bovis-derived ⁇ .
  • a plasmid pSBAA2 used to secrete amylase was constructed. [2-7] Preparation of plasmid used to display glucoamylase derived from Rhizopus oryzae ⁇ -amylase from Streptococcus bo vis as a fusion protein with the aggregation function domain on the cell surface
  • the prepro a-factor signal sequence gene was obtained as follows. Chromosome DNA of S. cer evisiae W303-IB was extracted. Using this as a template, PCR amplification was performed using 5, -AACGGAGCTCATGAGATTTCCTTCAATTTTTACTGCAGTT-3 '(SEQ ID NO: 9) and 5, -GGGGTACCGCATGCTCTTTTATCCAAAGATACCCCTTCTTCTTT-3' (SEQ ID NO: 10) as primers. Amplify 3 &. After digestion with 1 ⁇ 113111, a SacI-KpnI fragment of a prepro-factor signal sequence of about 250 b ⁇ in length was obtained.
  • [2-7-2] Acquisition of the gene for the 5 region (aggregating functional domain) of FLO 1 The gene for the 5 'region (aggregating functional domain) of FLO 1 was obtained as follows. First, chromosomal DNA was extracted from S. cerevisiae ATCC60715. Then, using this as a template, PCR amplification was performed using 5'-AGGAGGATCCGAGGCGTGC TTACCAGCAGGCCAGAGGAAA-3 '(SEQ ID NO: 11) and 5, -GCGAGTCGACTTMGATCT GGTGATTTGTCCTGAAGATGATGATGACAAA-3' (SEQ ID NO: 12) as primers, and the amplified product was BamH.
  • the plasmid pGA11 containing the darcoamylase gene was used as a template, and 5'-CGTTGGATCCGCAAGCATTCCTAGT AGTGCTTCTGTCCAG-3, (SEQ ID NO: 13) and 5'-ATCGGGATCCAGCGGCAGGTGCACC AGCCTTAGCGTA-3 '(SEQ ID NO: 14) were used as primers.
  • the amplified product was digested with BamHI to obtain an approximately 1800 bp dalcoamylase gene BamHI fragment. This fragment encoded the mature protein sequence excluding the secretory signal sequence of dalcoamylase.
  • the ⁇ -amylase gene of Streptococcus bovis was obtained as follows. Briefly, first, plasmid pQE31 :: amyA containing the ⁇ -amylase gene was used as a template, and 5,-CGTTAGATCTGAT GAACAAGTGTCAATGAAAGATGGTACG-3 '(SEQ ID NO: 15) and 5'-ATMCTCGAGTTA TTTTAGCCCATCTTTATTATAGTTTCC- PCR amplification was performed using 3 ′ (SEQ ID NO: 16), and the amplified product was digested with Bg1II and XhoI to obtain an approximately 2000 bp ⁇ -amylase gene Bg1II-XhoI fragment. Obtained. This fragment encoded the mature protein sequence except for the secretory signal sequence of the heat amylase.
  • plasmid pUC119 was digested with SacI and Kp ⁇ I, the SacI-KpnI fragment of the prepro a-factor signal sequence obtained in [2-7-1] was inserted. Then, plasmid; UC119 ⁇ was obtained. This plasmid pUC119 ⁇ was digested with BamHI and Sa1I, and the BamHI_Sa1I fragment of the FLO1 gene obtained in [2-7-2] above was obtained. And insert p UC 1 19 a FS was obtained.
  • the plasmid was inserted into the yeast constitutive expression plasmid pWGP3 digested with, and the yeast surface display plasmid pWGaFS was obtained.
  • the monoamylase gene Bg obtained in [2-7-4] above is digested.
  • the 1II-XhoI fragment was inserted to obtain a plasmid for surface display of human amylase pWGaFSA.
  • ⁇ -amylase is expressed as a fusion protein fused to the C-terminal side of the FLOl aggregation functional domain.
  • the plasmid pWGaFSA obtained in [2-7-6] is digested with BamHI, dephosphorylated, and the dalcoamylase gene BamHI fragment obtained in [2-7-3] is digested.
  • a plasmid pWGaGF S plasmid for displaying dalcoamylase and ⁇ -amylase as a fusion protein on the cell surface was obtained. Dalcoamylase and ⁇ -amylase are expressed as fusion proteins fused to the C-terminal side of the FLOL aggregation functional domain.
  • the plasmid pWGaFS for yeast surface display obtained in [2-7-5] is digested with BamHI, dephosphorylated, and the darcoamylase gene B a obtained in [2-7-3] is obtained.
  • the mHI fragment was inserted to obtain a plasmid pWGaGFS for display on the surface of Dalcoamylase.
  • the darcoamylase is expressed as a fusion protein fused to the N-terminal side of the FLO1 aggregation functional domain.
  • Example 1 Preparation of a flocculant yeast which presents dalcoamylase derived from Rhizopus oryzae on the cell surface by means of a GPI anchor and secretes human amylase derived from Bacillus stearothermophilus.
  • Plasmids for displaying dalcoamylase from Rhizopus oryzae on the cell surface with a GPI anchor: GAl1 and a plasmid for secreting ⁇ -amylase from Bacillus stearothermophilus; SAA1 1 and Yeast Maker (Clontech Laboratories , Inc., Palo Alto, Calif.) was simultaneously introduced into yeast YF207 by the lithium acetate method.
  • SD agar medium (6.7 g / l Yeast nitrogen base w / o amino acids (Difco Laboratories)) supplemented with appropriate amino acids and bases not containing L-tryptophan or uracil, 2 % Glucose, 0.02 g / l adenine sulfate, 0.02 g / 1 L-histidine / 1 ⁇ 1, 0.03 g / l L-leucine, 0.02 g / l L-lysine).
  • the grown yeast was selected and named as YF207-pGAl1 + PSAA11.
  • Plasmid pSGA11 for secreting dalcoamylase and plasmid pAAl2 for displaying ⁇ -amylase on the cell surface by GPI anchor were simultaneously introduced into yeast YF207 in the same manner as in Example 1.
  • the obtained yeast was designated as YF20 ⁇ / v SGA11 + pAA12.
  • Plasmid pGAl1 was transferred to yeast YF207 in the same manner as in Example 1 ', except that plasmid pGAl1 for presenting darcoamylase derived from Rhizopus oryzae on the cell surface was used, and peracyl lOg / 1 was added to the selection medium.
  • the yeast thus introduced and obtained was named YF207 / pGAl1.
  • Example 1 Each 5 ml of the transformed yeast obtained in Example 1 and Comparative Examples 1 to 3 was inoculated into 100 ml of SD medium containing 1% casamino acid (manufactured by Difco Laboratories), and shaken at 30 ° C. for 48 hours. Thus, seed culture was performed.
  • each seed culture was added to 1 L of 4% YPS medium (10 g / l Og / 1 yeast extra Tato (manufactured by Dco Laboratories), 20 g / l polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 40 g / l starch (soluble) (manufactured by Wako Pure Chemical Industries, Ltd.), 5 g / l glucose were pre-filled.
  • YPS medium 10 g / l Og / 1 yeast extra Tato (manufactured by Dco Laboratories), 20 g / l polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 40 g / l starch (soluble) (manufactured by Wako Pure Chemical Industries, Ltd.), 5 g / l glucose were pre-filled.
  • YPS medium 10 g / l Og / 1 yeast extra Tato (manufactured by Dco Laboratories),
  • the pH of the medium was maintained at 5.0 by the addition of sodium sulfate sulfate, and the dissolved oxygen concentration (DO) was maintained at 2.0 ppm by adjusting the stirring speed. After the weight of the dried cells reached about 15 g / l, the medium was removed, and the cells were collected by centrifugation at 5000 rpm for 10 minutes.
  • the yeast strain YF207 which is a host of the yeast used, hardly grew in this medium.
  • each of the collected yeast pellets is inoculated into 1 L of 6% YPS medium (that is, containing 60 g / l starch) in a jar arm mentor, and subjected to anaerobic conditions at pH 5.0 and 30 ° C. Fermentation was carried out for about 35 hours with gentle stirring (150 rpm) underneath. Throughout the cultivation and fermentation steps, starch concentration, dry cell weight, and ethanol concentration were determined.
  • the starch concentration was measured as follows. Cells were separated from the 1.0 ml sample by centrifugation at 5000 rpm for 5 minutes, and the supernatant was diluted with distilled water and used for starch concentration measurement. From Aspergillus niger of Darco amylase solution (6100 units / m l, Sigma Chemical Co., St. Louis, M0) were diluted 100-fold with distilled water, Darco amylase solution 0. lml the diluted sample of 0. 9 ml In addition, it was incubated at 30 ° C for 30 minutes. After the reaction was stopped by boiling for 10 minutes, the glucose concentration in the solution was measured using a glucose CII Test Co. (Wako Pure Chemical Industries, Ltd.) and a spectrophotometer (U-2001, Hitachi). And converted to starch concentration.
  • Aspergillus niger of Darco amylase solution (6100 units / m l, Sigma Chemical Co., St. Louis, M0) were diluted 100-fold with
  • the ethanol concentration was measured using a gas chromatograph equipped with a flame ionization detector.
  • the measurement was performed using 24 graphs (Model GC-8; manufactured by Shimadzu Corporation).
  • the measurement conditions were as follows: column, 3.0 side x 3.30 lm of Unisole 300 0 (GL Science Inc.) packed in glass; column temperature, 210 ° C; injector / detector temperature, 270 ° C; carrier gas, nitrogen (flow rate: 25 ml / min).
  • Table 1 shows the results of alcohol fermentation by cultivation and fed-batch culture of various yeasts.
  • feed l and feed 2 indicate the amount of starch (g) added after the measurement of the starch concentration at 36 hours and 72 hours, respectively.
  • the yeast of the present invention which presents dalcoamylase on the cell surface by means of a GPI anchor and secretes ⁇ - amylase, did not differ greatly from other yeasts in the growth phase.
  • the capacity was found to be superior to other yeasts (Example 2).
  • the alcohol production of the yeast of the present invention was about 77 g / l, which means that about 40% of the added starch was converted to alcohol. This value proved to be practical, comparable to the case where glucose was used as a substrate.
  • the alcohol yield of other yeasts is 34% for yeast that presents glucoamylase and ⁇ -amylase on the cell surface using a GPI anchor, and dalcoamylase is that when ⁇ -amylase is presented on the cell surface using a GPI anchor.
  • dalcoamylase is that when ⁇ -amylase is presented on the cell surface using a GPI anchor.
  • Example 3 Preparation of flocculent yeast which presents dalcoamylase derived from Rhizopus oryzae on the cell surface with a GPI anchor and secretes Streptococcus bovis-derived human amylase
  • the plasmid pGAl1i for displaying Dalcoamylase derived from Rhizopus oryzae on the cell surface with a GPI anchor and the plasmid pSBAA2 for secreting human amylase derived from Streptococcus bovis were used in the same manner as in Example 1 to obtain yeast YF.
  • the resulting yeast was named YF207 / [pGA11, SBAA2].
  • a plasmid pGAl1 for displaying Rhizopus oryzae-derived darcoamylase on the cell surface with a GPI anchor and a plasmid pBAAl for displaying ⁇ -amylase from Streptococcus bovis with a GPI anchor on the cell surface were prepared in Examples.
  • the yeast was simultaneously introduced into yeast YF207 in the same manner as in 1, and the resulting yeast was named YF207Z [pGAl1, BAA1].
  • Plasmid pBAAl for displaying Streptococcus bovis-derived CK monoamylase on the cell surface with a GPI anchor was introduced into yeast YF207 in the same manner as in Example 1, and the obtained yeast was named YF207 / pBAA1.
  • Plasmid P SBAA2 for secreting ⁇ -amylase derived from Streptococcus bovis was introduced into yeast YF207 in the same manner as in Example 1, and the obtained yeast was named YF207 / pSBAA2.
  • Example 3 For the yeasts obtained in Example 3 and Comparative Examples 7 to 9, expression of the cell surface of dalcoamylase by the plasmid pGAl1 was confirmed by flow cytometry analysis. The ⁇ -amylase activity of secretory expression by SBAA2 was confirmed by blue starch plate.
  • YF207 / [pGA11, pBAAl] co-presenting 29 ⁇ -amylase also grew well on steamed corn starch. These two types of recombinant yeast also consumed a large amount of starch in the culture medium after culturing for 48 hours.
  • Example 5 Preparation of non-aggregating yeast that presents Dalcoamylase from Rizopus oryzae and ⁇ -amylase from Streptococcus bovis as a fusion protein with an aggregation function domain on the cell surface
  • the plasmid pWG ⁇ GFSA obtained in [2-7-7] of Reference Example 2 above was non-aggregated yeast S. by the lithium acetate method using Yeast Maker (Clontech Laboratories, Inc., Palo Alto, CA). cerevisiae MT8-1 was introduced. This was purified using SD-W agar selection medium (6.7% yeast nitrogen base w / o amino acids (Difc Laboratories), 2% glucose, 2% agar powder supplemented with appropriate amino acids and bases without L-tryptophan). ). The grown yeast was selected, and the obtained transformant was named MT8-lZpWGaGFSA (Dalcoamylase + ⁇ -amylase fusion surface display yeast).
  • the plasmid pWGaGFS for displaying the Rhizopus oryzae-derived glycoamylase obtained in [2-8] of Reference Example 2 above on the cell surface was introduced into the non-aggregating yeast MT 8-1 in the same manner as in Example 5.
  • the obtained yeast was designated as MT 8-1 / pWG aGFS (Dulcoamylase surface-displaying yeast).
  • Comparative Example 15 Preparation of flocculant yeast that presents ⁇ -amylase derived from Streptococcus bovis on the cell surface by a flocculant domain) 8234
  • 31-Plasmid pWGaFSA for displaying Streptococcus bovis-derived CK monoamylase obtained in [2-7-6] of Reference Example 2 above on the cell surface was added to non-aggregating yeast MT8 in the same manner as in Example 5.
  • the resulting yeast was named MT 8-1 / p WGaF SA ( ⁇ -amylase surface display yeast).
  • a plate assay for dalcoamylase activity was performed as follows. After streaking each transformant on a plate medium consisting of 1% yeast extract, 2% peptone, 3% soluble starch, 0.003% promocresol purple, 0.5% glucose, and 2% agar powder, streak at 30 ° C. For 2 days and then at 4 ° C for 2 days. Glucoamylase activity was detected by observing a clear halo formed around the colony. The result is shown in FIG.
  • Dalcoamylase Surface-displaying yeast MT8-1 / pWGaGFS and Darcoamylase + ⁇ -amylase fusion surface-displaying yeast ⁇ 8-1 / pWGaGFSA was found to have sufficient dalcoamylase activity.
  • yeast cells displaying ⁇ -amylase on the surface of MT8-1 / pWG and FSA showed faint mouth formation.
  • a plate assay for ⁇ -amylase activity was performed as follows. Each transformant was streaked on a plate medium consisting of 1% yeast extract, 2% peptone, 0.25% remazole starch, 2% glucose, and 2% agar powder, and then cultured at 30 ° C for 4 days. Observing a clear halo formed around the colony As a result, ⁇ -amylase activity was detected. The result is shown in FIG.
  • ⁇ -amylase surface-displaying yeast ⁇ 8-1 / pWGaFSA and glucoamylase + ⁇ -amylase fusion surface-displaying yeast MT8-lZpWGoiGFSA It can be seen that it has an ⁇ -amylase activity. Also, as can be seen from the size of the mouth, the yeast displaying the surface of the dalcoamylase + ⁇ - amylase fusion surface was higher in activity than the yeast displaying the surface of the ⁇ - amylase. This indicates that fusion expression of the two amylases is useful.
  • 200 ml of the transformed yeast obtained in Example 5 was inoculated into 100 ml of YSP medium containing 5 g / l D-glucose (10 g / l yeast extract, 20 g / l polypeptone, 20 g / l steamed corn starch).
  • the yeast cells are grown and grown at 30 ° C with shaking under aerobic conditions for 48 hours, consuming starch and producing ethanol.
  • the yeast of the present invention which presents dalcoamylase on the surface and secretes ⁇ -amylase, is a yeast which presents dalcoamylase and ⁇ -amylase on the cell surface, respectively, when grown using starch as a substrate.
  • Yeast that presents dalcoamylase on the surface and secretes glucoamylase, and yeast that presents glucoamylase to cells are not much different, but have higher alcohol fermentation ability from starch than other yeasts. Since the alcohol yield is 40%, which is comparable to alcohol fermentation from Darcos, it is useful for producing ethanol directly from starch.
  • ⁇ -amylase derived from Streptococcus bovis when used for secretion, it is possible to use uncooked starch as a carbon source. Can also produce alcohol in high yield. Therefore, it is possible to produce ethanol more efficiently without cooking raw starch.
  • dalcoamylase + ⁇ -amylase fusion surface-displaying yeast also has the ability to efficiently ferment starch with alcohol, and is therefore useful for direct ethanol production from starch.

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Abstract

Selon cette invention, un plasmide construit de façon à présenter de la glucoamylase sur sa couche superficielle et à sécréter de l'α-amylase est intégré dans une levure qui fermente ensuite avec de l'amidon pour substrat. Ainsi, un alcool peut être efficacement produit par fermentation directement à partir de l'amidon. Cette levure est supérieure au niveau de sa capacité de fermentation alcoolique à une levure possédant de la glucoamylase et de l'α-amylase sur sa couche superficielle cellulaire, à une levure possédant de l'α-amylase sur sa couche superficielle cellulaire et sécrétant de la glucoamylase, et à une levure possédant de la glucoamylase sur sa couche superficielle cellulaire. De plus, à l'aide de l'α-amylase provenant du Streptococcus bovis, de l'éthanol peut être produit directement à partir d'amidon non cuit. Il est également possible d'utiliser une levure qui présente sur sa couche superficielle cellulaire de la glucoamylase et de l'α-amylase en tant que protéine hybride avec le domaine de fonction d'agglutination d'une protéine de liaison de chaîne glucidique.
PCT/JP2002/008234 2001-08-16 2002-08-12 Procede de production d'alcool a partir d'amidon Ceased WO2003016525A1 (fr)

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JP5626854B2 (ja) * 2010-06-08 2014-11-19 国立大学法人神戸大学 エタノールの生産方法
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