WO2003016525A9 - Process for producing alcohol from starch - Google Patents

Abstract

A plasmid having been constructed so as to present glucoamylase on its surface layer and secret α-amylase is integrated into a yeast and then the yeast is grown using starch as a substrate. Thus, al alcohol can be efficiently produced by fermentation directly from the starch. This yeast is superior in alcohol fermentability to a yeast presenting glucoamylase and α-amylase on the cell surface layer, a yeast presenting α-amylase on the cell surface layer and secreting glucoamylase, and a yeast presenting glucoamylase on the surface layer. Using α-amylase originating in Streptococcus bovis, moreover, ethanol can be directly produced from uncooked starch. It is also possible to employ a yeast which presents on the cell surface layer glucoamylase and α-amylase as a fused protein with the agglutination function domain of a sugar chain-binding protein.

Description

 Description Method for producing alcohol from starch

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 _α -amylase. Alternatively, the present invention relates to a method for producing ethanol using yeast that displays dalcoamylase and α-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

 The use of 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. In particular, 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 example of introducing a gene encoding a saccharified amylase (Dulcoamylase) into yeast to secrete saccharified amylase, growing this yeast using starch as the sole carbon source, and attempting alcohol fermentation. Yes (Nakamura et al. Biotechnol. Bioeng., 53: 21-25 (1997)). However, the growth of the yeast was not good, and the ethanol production was low, making it impractical. Furthermore, an attempt was made to immobilize dalcoamylase on the cell surface of yeast, grow this yeast using starch as the sole carbon source, and produce ethanol, and succeeded in increasing ethanol production (Ueda Appl. Environ. Microbiol., 63: 13 62-1366 (1997)) has not yet been put to practical use. Disclosure of the invention

 Therefore, 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.

As a result of various studies on efficient production of ethanol from starch, the present inventors transformed yeast to display dalcoamylase on the cell surface and to secrete or display _α -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 α-amylase.

 In a preferred embodiment, the dalcoamylase is presented via a GPI anchor.

 In a preferred embodiment, 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 α-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 GΡI 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.

 In a preferred embodiment, the sugar chain binding protein domain is a portion containing at least an aggregation functional domain of a GP I anchor protein.

 In a more preferred embodiment, the GPI anchor protein is an aggregated protein.

 In a further preferred embodiment, the aggregated protein is a protein selected from the group consisting of FL01, FL02, FL04, FL05, FLO9, FLO10, and FLO11.

 In another preferred embodiment, the α-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.

 In a preferred embodiment, the starch is uncooked. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram showing the construction of a plasmid pAAl2 that presents α-amylase on the cell surface by a GPI anchor.

 FIG. 2 is a schematic diagram showing the construction of plasmid pSAA11 that secretes α-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 | concentration in a culture medium, and a concentration of ethanol. 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 α-amylase activity. BEST MODE FOR CARRYING OUT THE INVENTION

 As used herein, 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. For example, darcoamylase derived from molds such as Rhizopus and Aspergillus is used. For example, as described in Ueda et al. (Appl. Environ. Microbiol. 63: 1362-1366 (1997)), darcoamylase derived from Rhizopu s oryzae is preferably used.

In the present invention, α-amylase refers to an endo-type enzyme that hydrolyzes α1,4-darcoside bonds of starch. The source of the activity is not limited as long as it has this activity. For example, α-amylase derived from animals (such as saliva and kidney), plants (such as malt), and microorganisms is used. In the present invention, alpha-amylase force derived from a microorganism ^s preferred [this for Rere is, for example ho ,, Bacillus stearothermophi lus, include those such as from Streptococcus bovis. As described later, α-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 α-amylase. Such a yeast can be obtained by introducing into a yeast D Ν 組 換 え recombined to display glucoamylase on the cell surface and D Ν 組 換 え recombined to secrete α-amylase. , can get.

 The second yeast of the present invention is a yeast transformed to display dalcoamylase and α-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, α-amylase, and carbohydrate-binding protein domains as a single fusion protein on the cell surface. Can be

 First, a general method for presenting an enzyme on the cell surface will be described. 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. There is a method in which an enzyme is displayed on the cell surface through a chain binding protein domain. When one type of enzyme is displayed on the cell surface, any of the methods (a) and (b) may be used. When two types of enzymes are displayed on the cell surface, it is preferable to express the fusion protein by the method (b). Cell surface localization proteins that can be used include yeast sex aggregation proteins α- or a-agglutune, FLO proteins (eg, FL01, FLO2, FL04, FL05, FL09, FLO10, and FLOl 1), alkaline phosphatase And the like.

 (a) How to use GPI anchor

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.

 Here, the GPI anchor refers to a glycosylphosphatidylinositol (GPI) ethanolamine phosphate-6 mannose α 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. As the GPI anchor attachment signal sequence, for example, the sequence of the C-terminal part of yeast agglutin is preferably used. On the C-terminal side of the sequence of C-terminal 320 amino acids from the C-terminal of the α-agglutene, a GPI anchor attachment recognition signal sequence is included. DNA sequences encoding are particularly useful.

Therefore, for example, in the sequence having a DNA sequence encoding a secretion signal sequence and a DNA sequence encoding a GPI anchor attachment recognition signal, 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. Cell surface localization protein T / JP02 / 08234

In the case of 7-glutinin, it is preferable to introduce a target enzyme gene so as to leave a sequence encoding a sequence of 320 amino acids from the C-terminal of the _α -agglutinin.

 When 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.

 (b) Method using a sugar chain binding protein domain

 When the cell surface localized protein is a sugar chain-binding protein, 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. For example, 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. Depending on the type of the target enzyme, (1) the enzyme is bound to the N-terminal side of the cell surface localized protein (aggregation functional domain), (2) the enzyme is bound to the C-terminal side, and (3) the N-terminal side. The same or different enzymes can be bound to both the C-terminal side and the C-terminal side.

 So, for example,

 (1) 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)

(2) 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;

(3) DNA encoding secretory signal sequence—Structural gene of target enzyme Structural gene coding for one cell surface localization protein (aggregation function domain) 1 Structural gene of another enzyme of interest, etc. can get. When using an aggregation domain, the GPI anchor is not involved in the display of the cell surface, so the DNA sequence encoding the GPI anchor attachment recognition signal sequence may or may not be present in the recombinant DNA. You don't have to.

 When the dalcoamylase gene is used as the structural gene of the desired enzyme, a recombinant DNA that displays dalcoamylase on the cell surface using the sugar chain binding protein domain can be obtained. Alternatively, recombinant DNΑ having a dalcoamylase structural gene and an α-amylase structural gene at the N-terminal and C-terminal of the sugar chain-binding protein domain, respectively, were obtained, and both enzymes were expressed as fusion proteins on the cell surface. You can also.

 As the secretory signal sequence used in the above-mentioned recombinant DNA, 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. For example, a secretory signal sequence for dalcoamylase, a secretory signal sequence for yeast α- 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.

Next, methods for allowing yeast to secrete amylases (such as dalcoamylase and α-amylase) are well known to those skilled in the art. A recombinant DN DN in which a structural gene of a target enzyme such as dalcoamylase or _α -amylase is linked to DNA encoding the above-mentioned secretory signal sequence may be introduced into yeast.

The synthesis and binding of 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 (recombinant DNA) is preferably incorporated into a vector. From the viewpoint of facilitating DNA acquisition, a shuttle vector with Escherichia coli is preferable. For example, 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. More preferably, it has a selection marker (eg, drug resistance gene, TRP, LEU2, etc.) and an E. coli selection marker (eg, drug resistance gene).

 In order to express the dalcoamylase or α-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. For example, 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 And a plasmid pWI3 containing a Term-ICL (terminator region of isocitrate lyase) sequence.

 When presenting the enzyme on the cell surface of yeast, most preferably, 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 α-amylase structural gene and the sequence encoding 320 amino acids from the C-terminus of α-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

 10 Each may be integrated into a different vector.

 As the yeast of the host, 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.

 Examples of the yeast of flocculation are 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 α-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 α-amylase, or a yeast which presents dalcoamylase and α-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 DNΑ 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 α-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 α-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. For the immobilization of yeast, 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

 Hereinafter, a method for producing ethanol by fermenting the yeast of the present invention in the presence of starch will be described.

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. When uncooked starch is used, 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.

 Next, 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. In the fed-batch batch process, 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.

In the case of a batch (batch, fed-batch, repetitive batch) process, the starch concentration in the medium is preferably about 40 to about 150 g / l. In particular, the starch concentration is more preferably from about 50 to about 120 g / l, most preferably about 60 g / l. Also, in the case of a continuous process, 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. When non-steamed starch is used, 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.溶 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 ρρη. 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.

 Since the above fermentation conditions change as the fermentation progresses, it is preferable to adjust these to a certain range. Changes over time in the fermentation can be monitored by those skilled in the art, such as gas chromatography and HPLC, using ordinary methods.

 During or after the fermentation step, 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.

 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples. Example

 Reference example Preparation of experimental materials

 (Reference Example 1 Breeding of host yeast)

 Saccharomyces diastaticus ATCC60712 (MATa leu2-3, 1 12 his2 lys2 stal FL08), which is an aggregating yeast, and W303-IB (ΜΑΤ α 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 Δ 2 his ade2-1 canl-). 100 stal FL08).

In addition, the non-aggregating yeast Saccharomyces cerevisiae MT 8-1 (MATa ade his3 leu2 trpl ura3) (Tajima et al., Yeast, 1: 67-77, 1985) Used as yeast.

(Reference Example 2 Preparation of various plasmids used in Examples and Comparative Examples)

 [2-1] Preparation of plasmid used to display dalcoamylase from Rhizopus oryzae on cell surface by GPI anchor

 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.

[2-2] Preparation of plasmid used to display a-amylase from Bacillus stearothermophilus on cell surface by GPI anchor

 Plasmid p AA12, which presents α-amylase from Bacillus stearothermophilus on the cell surface, was prepared using plasmid ρIΑΑΔ11 as a starting material. Figure 1 shows a schematic diagram of the construction. Plasmid ρ Ι ΑΑΔ11 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 α-agglutin. On the other hand, the multicopy plasmid ΡΜΤ34 (+3) was digested with ΡVuII 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 ΑΑΔ11 with Xho I was used to obtain a secretion signal sequence of about 2,000 b of the yeast a-functional enzyme. _Α -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 α-amylase on the cell surface. [2-3] Preparation of plasmid used to secrete α-amylase from Bacillus stearothermophilus

 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 α-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). Prepared by Figure 2 shows a schematic diagram of the construction. : Perform PCR using IAA11 as a template, 5'-ATCGCGAGCTCATGAGATTTCCTCCA ATTTTTACTGCAG-3 '(SEQ ID NO: 1) and 5'-ATGCGAGCTCTCAAGGCCATGCCACCAAC CGTGGTTCGG-3' (SEQ ID NO: 2) as primers and cut with SacI As a result, a fragment having a length of about 2000 bp was obtained. This fragment encoded the secretory signal sequence of yeast α-fatty acid and the mature protein sequence of α-amylase. The obtained sequence was ligated to pUGP3 previously treated with SacI to obtain a plasmid pSAA11.

[2-4] Preparation of plasmid used to secrete darcoamylase from Rhizopus oryzae

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. On the other hand, 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.

[2-5] Preparation of plasmid used to display human amylase from Streptococcus bovis on cell surface by GPI anchor

 Plasmid pBAAl which presents α-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 α-agglutinin. On the other hand, the multicopy plasmid ρΜΤ34 (+3) was digested with ΡVuII and BamHI, blunted, and the above fragment of about 860 bp was incorporated to obtain plasmid pUGP12. Was.

Plus! A plasmid in which an α-amylase gene derived from Streptococcus bovis 418 (E. Satoh et al., Appl. Environ. Microbiol. 63: 4593-4596 (1997)) was inserted into QE31 (Qiagen)! ) QE31 :: AmyA was created. This was digested with Sac II and Xho I to obtain a single amino acid from S. bovis 418. A fragment containing the lase gene was obtained. Next, plasmid pCASl (S. Shibasaki et al., Appl. Microbiol. Biotechnol. 55: 471-475 (2001)) was digested with SacII and XhoI, and α-derived from S. bovis 418 was added thereto. —Amylase gene fragment was inserted to obtain plasmid pCAS1 :: amyA. 5'-AATACTCGAGATGCAACTGTTCA using this plasmid pCAS1 :: amyA as a template

ATTTGCCATTGAAAGT-3, (SEQ ID NO: 5) and 5, -CTGCCCATGGGGTTTTAGCCCATCTT TATTATAGTTTCC-3 '(SEQ ID NO: 6) Amplified by PCR, treated with Xhol, and secreted signal sequence of dalcoamylase gene. A 2.2 kb DNA fragment containing the α-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 α-amylase derived from Streptococcus bovis on the cell surface.

[2-6] Preparation of plasmid used to secrete a-amylase from Streptococcus bovis

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. On the other hand, the multicopy-type plasmid pUGP3 (Takaha shi et al., Supra) was digested with restriction enzymes SacI and KpnI, and the above SacI- nρnI fragment was ligated therewith, thereby obtaining Streptococcus bovis-derived α. A plasmid pSBAA2 used to secrete amylase was constructed. [2-7] Preparation of plasmid used to display glucoamylase derived from Rhizopus oryzae α-amylase from Streptococcus bo vis as a fusion protein with the aggregation function domain on the cell surface

 [2-7-1] Acquisition of prepro α-factor signal sequence gene

 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 ぉ 113111, a SacI-KpnI fragment of a prepro-factor signal sequence of about 250 bρ 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. Digestion with I and Sa1I resulted in a BamHI-Sa1I fragment of approximately 3300 bj) in length. This fragment encodes the FLO1 aggregation functional domain except for the secretory signal sequence on the 5 'side of FLO1, and has the sequence described in Watari et al. (Yeast 10 (2): 211-25 (1994)). Matches [2-7-3] Acquisition of darcoamylase gene of Rhizopus oryzae The darcoamylase gene of Rhizopus oryzae was acquired as follows. Briefly, first, 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.

[2-7-4] Acquisition of α-amylase gene of Streptococcus bovis

 The α-amylase gene of Streptococcus bovis was obtained as follows. Briefly, first, plasmid pQE31 :: amyA containing the α-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 α-amylase gene Bg1II-XhoI fragment. Obtained. This fragment encoded the mature protein sequence except for the secretory signal sequence of the heat amylase.

[2-7-5] Preparation of plasmid for yeast surface display

First, after cloning plasmid pUC119 was digested with SacI and KpηI, the SacI-KpnI fragment of the prepro a-factor signal sequence obtained in [2-7-1] was inserted. Then, plasmid; UC119α was obtained. This plasmid pUC119α 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 p UC 1 1 9 o; was digested FS in S acl and S a 1 I, the _a FS gene S ac I -S ail fragment obtained about 3500 bp, S ac I and S a 1 I The plasmid was inserted into the yeast constitutive expression plasmid pWGP3 digested with, and the yeast surface display plasmid pWGaFS was obtained.

[2-7-6] Preparation of plasmid used to display α-amylase from Streptococcus bovis on cell surface by using aggregation domain

 After digesting the plasmid pWGaFS for yeast surface display obtained in [2-7-5] above with Bg1II and XhoI, 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. Α-amylase is expressed as a fusion protein fused to the C-terminal side of the FLOl aggregation functional domain.

[2-7-7] Glucoamylase from Rhizopus oryzae Preparation of plasmid used to display a single amylase from Streptococcus bovis as a fusion protein on cell surface

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. By insertion, a plasmid pWGaGF S plasmid for displaying dalcoamylase and α-amylase as a fusion protein on the cell surface was obtained. Dalcoamylase and α-amylase are expressed as fusion proteins fused to the C-terminal side of the FLOL aggregation functional domain. [2-8] Preparation of plasmid used to display dalcoamylase from Rhizopus oryzae on the cell surface by using the 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 α-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. This was used as a selective medium, 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 / 1Κ1, 0.03 g / l L-leucine, 0.02 g / l L-lysine). The grown yeast was selected and named as YF207-pGAl1 + PSAA11.

(Comparative Example 1: Preparation of flocculent yeast that presents Dalcoamylase derived from Rhizopus oryzae and α-amylase derived from Bacillus stearothermophilus to the cell surface using a GPI anchor, respectively) Plasmid for displaying Rhizopus oryzae-derived darcoamylase on the cell surface with a GPI anchor: GAl1 and plasmid pAAl2 for displaying Bacillus stearothermophilus-derived α-amylase on the cell surface in the same manner as in Example 1. At the same time, and the resulting yeast was named YF207 / pGAl1 + pAA12.

(Comparative Example 2: Preparation of yeast that secretes darcoamylase derived from Rhizopus oryzae and displays α-amylase derived from Bacil lus stearothermophilus on the cell surface using a GPI anchor)

 Plasmid pSGA11 for secreting dalcoamylase and plasmid pAAl2 for displaying α-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 YF20Ί / v SGA11 + pAA12. (Comparative Example 3: Preparation of flocculent yeast displaying Dalcoamylase from Rhizopus oryzae on the cell surface by GPI anchor)

 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 2, Comparative Examples 4 to 6)

 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.

Then, 50 ml of 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. Each was placed in a 2 L jar armmenter (BMJ-02PI, Biott Corp., Tokyo) and cultured at 30 ° C under aerobic conditions. 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.

 Using the obtained yeast strain, batch fermentation was performed to produce ethanol. That is, 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.

The ethanol concentration was measured using a gas chromatograph equipped with a flame ionization detector. PC Cough 34

 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. In Table 1, 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. Means the starch concentration (g / 1) in the medium.

IN3

 o

* 1 GA: glucoamylase * 3 (alcohol production amount added τ starch amount) X 100 * 2 AA: Hi-amylase '

Both the yeast of Example 1 and the yeasts of Comparative Examples 1 to 3 grew using starch as a carbon source and fermented with alcohol, because the enzymes presented on each surface layer and the secreted enzymes separated the starch from each other. Glucose was produced, indicating that it was used.

The yeast of the present invention, which presents dalcoamylase on the cell surface by means of a GPI anchor and secretes _α- amylase, did not differ greatly from other yeasts in the growth phase. The capacity was found to be superior to other yeasts (Example 2). In other words, it was found that, compared to other yeasts, the angular velocity of starch was faster, the amount of remaining starch was smaller, and the speed of alcohol fermentation was higher. 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 _α -amylase on the cell surface using a GPI anchor, and dalcoamylase is that when α-amylase is presented on the cell surface using a GPI anchor. Was 34% in yeast secreting, and 31% in yeast displaying dalcoamylase on the cell surface by GPI anchor. This indicates that the yeast of the present invention is suitable for performing alcohol fermentation directly from starch and has high practicality.

After completion of the reaction, the yeast of the present invention used in Example 3 was diluted with an SD medium containing no tributophan and peracil, and plated on a YPD plate and an SD plate containing no triptophan and peracil. After incubation for 48 hours at, the number of colonies on both plates was counted. As a result, 80% or more of the yeasts stably retained plasmid. (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. At the same time, the resulting yeast was named YF207 / [pGA11, SBAA2].

(Comparative Example 7: Preparation of cohesive yeast that presents gnorecoamylase from Rhizopus oryzae and α-amylase from Sreptococcus bovis on the cell surface using GPI anchors, respectively)

 A plasmid pGAl1 for displaying Rhizopus oryzae-derived darcoamylase on the cell surface with a GPI anchor and a plasmid pBAAl for displaying α-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].

(Comparative Example 8: Preparation of flocculant yeast that presents α-amylase derived from Streptococcus bovis on the cell surface by GPI anchor)

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. 8234

 28

(Comparative Example 9: Preparation of aggregating yeast secreting α-amylase from Streptococcus bovis)

 Plasmid P SBAA2 for secreting α-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.

 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 α-amylase activity of secretory expression by SBAA2 was confirmed by blue starch plate.

(Example 4, Comparative Examples 10 to 13)

 200 μl of each of the transformed yeasts obtained in Example 3, Comparative Examples 3 and 7 to 9 was added to 100 ml of YSP medium containing 5 g / 1 D-glucose (lOg / 1 yeast extract, 20 g / 1 polypeptone, 20 g / l steamed corn starch was inoculated, and the yeast cells were grown by shaking for 48 hours under aerobic conditions at 30 ° C. The results are shown in FIG.

Streptococcus bovis derived from α- amylase only both YF 207Zetarobetaarufaarufa1 and YF 207 / p SBAA2 presented or secreted to the cell surface by GPI anchor, can grow steaming corn starch as a carbon source, 0D ₆₀₀ with its after 48 hours Reached 14. On the other hand, the dalcoamylase-presenting yeast YF207 / pGAl1 grew only slightly.

YF207 / [pGAl1, pSBAA2], which presents Rhizopus oryzae-derived dalcoamylase on the cell surface with a GPI anchor and secretes Streptococcus bovis ο; -amylase, grows on steamed corn starch medium. 0D ₆ after 48 hours. . Reached 25. Dalcoamylase and 0208234

 YF207 / [pGA11, pBAAl] co-presenting 29α-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.

Next, we examined alcoholic fermentation using a non-steamed corn starch medium. Each of the above five types of recombinant yeast cells was cultured under aerobic conditions at 30 ° C for 48 hours in an SDC medium (6.7 g / l yeast nitrogen base, lOg / 1 casamino acid supplemented with appropriate amino acids and nucleotides). , 20 g / l glucose) and harvested by centrifugation at 500 OXg for 10 minutes. The resulting cell pellet, YPS medium 50 ml (lOg / 1 yeast extract, 20 g / l polypeptone, to 50 g / l uncooked corn starch, were inoculated in yeast cell concentration in the initial 0D ₆ .. = 60. Then, Alcohol fermentation was performed while stirring (100 rpm) under anaerobic conditions at 30 ° C. The results are shown in FIG.

 As can be seen from FIG. 7, when 50 g / l of uncooked cornstarch was used as a carbon source, YF207Z that presents Dalcoamylase from Rhizopus oryzae and secretes α-amylase from Streptocoecus bovis [: GA11,

 Only SBAA2] was able to consume starch and produce ethanol. On the other hand, other recombinant yeast cells produced very little ethanol and had little growth.

Therefore, the effect of the starch concentration on alcohol fermentation was examined using YF207ΖCpGAll, pSBAA2, which can produce ethanol from uncooked starch. The initial 0D _60. Was set to 60. As shown in Figure 8, the higher the starch concentration, the higher the ethanol concentration produced. In the case of 250g / l uncooked corn starch, the ethanol concentration reached 50g / l in about 80 hours.

 In addition, when using 250 g / l of non-steamed corn starch medium,

The cell concentration of F207 / [pGAl1, SBAA2] was examined. Figure Initial 0D ₆ as shown in 9. . As the concentration increased, the initial ethanol production rate increased, but the final ethanol concentration decreased. On the other hand, when the yeast cell concentration was reduced to initial 0D _60O = 30, the ethanol concentration reached 60 g / l, but by the time it reached 50 g / l ethanol, the initial OD ₆₀ . = It took longer than 60.

(Example 5: Preparation of non-aggregating yeast that presents Dalcoamylase from Rizopus oryzae and α-amylase from Streptococcus bovis as a fusion protein with an aggregation function domain on the cell surface)

The plasmid pWGαGFSA 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 + _α -amylase fusion surface display yeast).

(Comparative Example 14: Preparation of non-aggregating yeast that presents dalcoamylase derived from Rhizopus oryzae on the cell surface using an aggregation-functional domain)

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 α-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 (α-amylase surface display yeast).

(Example 6, Comparative Examples 16 to 17)

 For each of the transformed yeasts obtained in Example 5 and Comparative Examples 14 and 15, 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 + α-amylase fusion surface-displaying yeast ΜΤ 8-1 / pWGaGFSA Was found to have sufficient dalcoamylase activity. In addition, yeast cells displaying α-amylase on the surface of MT8-1 / pWG and FSA showed faint mouth formation.

(Example 7, Comparative Examples 18 to 19)

For each of the transformed yeasts obtained in Example 5 and Comparative Examples 14 and 15, a plate assay for α-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, α-amylase activity was detected. The result is shown in FIG. α-amylase surface-displaying yeast Τ 8-1 / pWGaFSA and glucoamylase + α-amylase fusion surface-displaying yeast MT8-lZpWGoiGFSA It can be seen that it has an α-amylase activity. Also, as can be seen from the size of the mouth, the yeast displaying the surface of the dalcoamylase + _α- amylase fusion surface was higher in activity than the yeast displaying the surface of the _α- amylase. This indicates that fusion expression of the two amylases is useful.

(Example 8)

 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.

Industrial applicability

The yeast of the present invention, which presents dalcoamylase on the surface and secretes α-amylase, is a yeast which presents dalcoamylase and α-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. In particular, when α-amylase derived from Streptococcus bovis is 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. In addition, dalcoamylase + α-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.

Claims

The scope of the claims 1. A yeast that displays dalcoamylase on the cell surface and secretes an amylase. 2. The yeast of claim 1, wherein said dalcoamylase is presented via a GPI anchor. 3. The yeast according to claim 1, wherein the dalcoamylase is presented as a fusion protein with a sugar chain binding protein domain having no GPI anchor attachment recognition signal sequence. 4. A yeast which presents dalcoamylase and α-amylase to the cell surface as a fusion protein with a sugar chain binding protein domain having no GPI anchor attachment recognition signal sequence, wherein the glucoamylase is located at one end of the domain. A yeast, wherein the α-amylase is fused to the other end. 5. The yeast according to claim 3, wherein the sugar chain binding protein domain is a portion containing at least an aggregation functional domain of a GPI anchor protein. 6. The cell surface binding protein according to claim 5, wherein the GPI anchor protein is an aggregated protein. 7. The cell surface-binding protein according to claim 6, wherein the aggregated protein is a protein selected from the group consisting of FL01, FL02, FL04, FL05, FL09, FLO10, and FLO11. 8. The yeast according to any one of claims 1 to 7, wherein the α-amylase is derived from Streptococcus bovis. 9. A method for producing alcohol, comprising a step of culturing the yeast according to any one of claims 1 to 8 in a medium containing starch. 10. The method of claim 9, wherein the starch is uncooked.