WO2002042483A1

WO2002042483A1 - Process for producing ethanol from starch

Info

Publication number: WO2002042483A1
Application number: PCT/JP2001/001538
Authority: WO
Inventors: Hideki Fukuda; Akihiko Kondo; Atsuo Tanaka; Mitsuyoshi Ueda; Hideo Noda; Moriyasu Abe; Hisayori Shigechi; Shouji Takahashi
Original assignee: Kansai Chemical Engineering Co., Ltd.
Priority date: 2000-11-27
Filing date: 2001-02-28
Publication date: 2002-05-30
Also published as: JP4666884B2; JPWO2002042483A1

Abstract

A yeast presenting glucoamylase on the surface layer of the cells; and an industrially applicable process for producing ethanol at a higher efficiency which comprises repeating a batchwise or continuous fermentation step by using this yeast.

Description

Description Method for producing ethanol 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 that displays dalcoamylase on the cell surface. 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 current fermentation processes is a two-step process: starch saccharification by steaming and enzymatic treatment, followed by yeast fermentation. This is because yeast does not have a secretory enzyme such as amylase, so that yeast cannot degrade starch to saccharify it and cannot use starch as a carbon source. Thus, in sake brewing, rice starch is sugar-dried using a koji mold or the like that secretes amylase, and then yeast is allowed to act on the starch to carry out alcohol fermentation to produce alcohol. Therefore, if a gene encoding amylase is introduced into yeast to secrete amylase, the yeast will grow using starch as the sole carbon source, and alcohol fermentation will be possible. Several such dalcoamylase-secreting yeasts have been constructed (Briol, G. et al., Enzyme Microb. Technol., 22: 672-677 (199). 8); Cole, GE et al., Bio / Technol., 6: 417-421 (1988); Ibragimova, SI et al., Biotechnol. Bioeng., 46: 285-290 (1995); Inlow, D. et al., Biotechnol. Bien. g., 32: 227-234 (1988); Innis, MA et al., Science 288: 21-26 (1985); Nakamura et al., Biotechnol. Bioeng., 53: 21-25 (1997)). However, in all cases, the growth of yeast and the production of ethanol are low and are not practical.

Thus, for example, immobilization on the cell surface of yeast would allow more efficient alcohol fermentation than secretion of amylase. The present researchers succeeded in immobilizing dalcoamylase on the surface of non-aggregating yeast, growing this yeast using starch as the sole carbon source, and producing alcohol (Ueda et al., Appl. Environ. Microbiol., 63: 1362-1366 (1997)). However, at this stage, industrial utilization was difficult due to the low stability of the plasmid incorporated into the yeast and the low yield of ethanol. Disclosure of the invention

Therefore, there is a need for a more efficient ethanol production process that can be used industrially. An object of the present invention is to provide a method for producing ethanol with higher efficiency by using a more stable yeast that presents dalcoamylase on the cell surface.

The present invention relates to a method for producing ethanol, which comprises a step of fermenting in the presence of starch using a flocculent yeast having DΝΑ recombined to display dalcoamylase on the cell surface.

In a preferred embodiment, the step of fermenting is a step of repeated batch fermentation or a step of continuous fermentation.

In a preferred embodiment, the fermentation step is performed in a medium containing 40 to 300 g / 1 of starch at pH 4 to 6 at 20 to 45 ° C under anaerobic conditions. In another preferred embodiment, in the continuous fermentation step, the supply rate of the medium is 0.07 to 0.2 vZv% Z hours of the amount of the medium.

In a preferred embodiment, the DNA is in the form of a plasmid, and the plasmid is a multicopy vector or a chromosomal integration vector.

In a preferred embodiment, the aggregating yeast is derived from the YF207 strain, and the aggregating yeast having the DNA is YF207 / pGA11 or YF207 / IGA11.

The present invention also includes a step of repeatedly performing batch fermentation or continuous fermentation in the presence of starch using a non-aggregating yeast having a DNA modified to present dalcoamylase on the cell surface. It relates to a manufacturing method. In a preferred embodiment, the fermentation step is performed in a medium containing 40-300 gZl of starch at pH 4-6 at 20-45 ° C under anaerobic conditions.

In a preferred embodiment, in the continuous fermentation step, the supply rate of the medium is 0.07 to 0.2 v / v% / hour of the amount of the medium.

In another preferred embodiment, the DNA is in the form of a plasmid, and the plasmid is a multicopy vector or a chromosomal integration vector. Furthermore, the present invention also relates to an aggregating yeast that presents dalcoamylase on the cell surface. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the structures of plasmid pGAl1 and plasmid: pIGA11.

Figure 2 shows the results of (a) in a batch fermentation process using YF207Zp GA11. Starch, glucose, and ethanol concentrations in the medium, and

(b) It is a graph which shows the time-dependent change of dry cell weight and dalcoamylase activity.

Figure 3 shows (a) starch concentration, glucose concentration, and ethanol concentration in the medium, as well as (b) dry cell weight, dalcoamylase activity, and the following in a repeated batch fermentation process using YF207 / pGAl1. 5 is a graph showing the change over time in the stability of plasmid.

Figure 4 shows (a) starch concentration, glucose concentration, and ethanol concentration in the culture medium, and (b) dry cell weight and dalcoamylase in the fed-batch fermentation process using YF207 / pGA11. It is a graph showing a time-dependent change in activity.

BEST MODE FOR CARRYING OUT THE INVENTION

The yeast used in the method of the present invention is a yeast transformed by introducing DNA so that dalcoamylase is displayed on the cell surface. The introduced DNA contains, in this order, a secretory signal sequence, a structural gene sequence for dalcoamylase, a sequence encoding a part of a cell surface localization protein, and a GPI anchor attachment signal sequence.

The secretory signal sequence is an amino acid sequence containing a large number of highly hydrophobic amino acids, which is bound to the N-terminus of proteins (secretory proteins) that are generally secreted extracellularly (including periplasm). It is removed when proteins are secreted from inside the cell, across the cell membrane and out of the cell.

In the present invention, any secretory signal sequence that can secrete (migrate) dalcoamylase out of yeast cells can be used, regardless of its origin. For example, the secretory signal sequence includes the secretory signal sequence of dalcoamylase and the yeast α- or a-agglutinin signal. And the like are preferably used. Some or all of the secretory signal sequence may remain at the N-terminus of the dalcoamylase provided that it does not affect the activity of the dalcoamylase.

The cell surface display protein is a protein that is fixed on the yeast cell surface and displayed on the cell surface. For example, _α- or a-adaltin, which is a sex aggregation protein, can be mentioned. Such proteins are similar to secreted proteins in having a secretory signal sequence, but differ from secreted proteins in that they are anchored and transported to the cell membrane via GPI anchors. The cell surface display protein has a GPI anchor attachment recognition signal sequence at the C-terminus, and its recognition signal sequence is fixed to the cell membrane by binding to the GPI anchor at the selectively cleaved C-terminal portion. You. Then, the base of the GPI anchor is cut by PI-PLC, incorporated into the cell wall, fixed to the cell surface, and presented to the cell surface.

Here, the GPI anchor is based on ethanolamine phosphate-6 mannose α 1-2 mannose a 1-6 mannose α 1-4 darcosamine α 1-6 inositol phospholipid called glycosylphosphatidylinositol (GPI) Refers to glycolipids having a structure. ΡΙ-PLC refers to phosphatidylinositol-dependent phospholipase C.

A sequence encoding a part of a cell surface localization protein is a sequence encoding a part of a protein (for example, or a-agglutinin) displayed on a yeast cell surface, and mainly a sequence encoding a C-terminal portion. However, any sequence may be used as long as it does not adversely affect the activity of glucoamylase. Preferably, a sequence encoding a sequence of 320 amino acids from the C-terminal of α-agglutinin is used. There are four carbohydrate binding sites in this amino acid sequence. After the GPI anchor is cleaved with ΡΙ-PLC, the sugar chain and the polysaccharides that make up the cell wall are covalently linked, and the C-terminal sequence of agglutin is linked to the cell wall. It is particularly useful because it is kept together.

The GPI anchor attachment signal sequence is a sequence recognized when the GPI anchor binds to a cell surface localized protein, and is usually a sequence located at or near the C-terminus of a cell surface localized protein. is there. A sequence encoding the C-terminal part of the yeast α-agglutinin sequence is preferably used. The sequence encoding the G アミノ酸 I anchor attachment signal sequence is included at the 3rd and 3rd ends of the sequence encoding the sequence of 320 amino acids from the C-terminus of α-agglutinin. A sequence encoding a sequence of 0 amino acids is useful.

As used herein, dalcoamylase refers to an exo-type hydrolase that separates a glucose unit from the non-reducing end of starch. As long as it has such activity, its origin does not matter, but fungal dalcoamylase such as Rhizopus and Aspergillus is used. For example, as described in Ueda et al. (Supra), darcoamylase derived from Rhizopus oryzae is preferably used. An unknown dalcoamylase gene may be determined and used by a person of ordinary skill in the art using a known method, or a known darcoamylase sequence is used.Secretion signal sequence, dalcoamylase structural gene sequence, cell surface localized protein Synthesis of a DNA comprising a sequence encoding a part of the sequence and a GPI anchor attachment signal sequence in this order is performed by a technique commonly used by those skilled in the art. For example, the binding between the secretory signal sequence and the structural gene of dalcoamylase can be performed using a site-directed mutagenesis method, which allows accurate cleavage of the secretory signal sequence and expression of active dalcoamylase. Furthermore, this sequence may be combined with a sequence encoding a part of a cell surface localization protein, such as a GPI anchor attachment signal sequence.

Preferably, the DNA is in the form of a plasmid. From the viewpoint of simplification of obtaining DNA, a shuttle vector with Escherichia coli is preferable. this Starting materials for DNA include, for example, a replication origin (0 ri) of yeast 2 μπι plasmid and a replication origin of ColEl, and a yeast selection marker (eg, drug resistance gene, TRP, LEU2, etc.). ) And a selection marker for E. coli (such as a drug resistance gene). In addition, in order to express the dalcoamylase structural gene, it is preferable to include a so-called regulatory sequence such as an operator that controls the expression of this gene, a motor, a terminator, and an enhancer. Examples include the GAPDH (daricelaldehyde 3'-phosphate dehydrogenase) promoter and the GAPDH terminator. Such starting material plasmids include pYE22m, pYGA2270, and the like.

Most preferably, between the GAPDH promoter and the sequence of the GAPDH terminator of the plasmid PYGA2270 or pYE22m, a sequence having a secretory signal sequence and a structural gene sequence of glucoamylase, and 320 C from the C-terminal of a-agglutinin. Insertion of a sequence linked to a sequence encoding an amino acid produces a plasmid used for introduction into yeast. In the present invention, the multicopy pGA11 and chromosomal integration pIGA11 thus produced are preferably used (FIG. 1).

As the yeast of the host, any yeast may be used as long as it has an alcohol fermentation ability. Non-agglutinating yeast is used. The cohesive yeast force S is preferable because it can be separated easily after the reaction, or because it can be fixed easily, so that a continuous reaction can be performed.

The non-aggregating yeast is not particularly limited, and examples thereof include Saccharomyces ce revisiae MT8-1.

Examples of the cohesive yeast include Saccharomyces diastaticus ATCC60715, ATCC60712, Saccharomyces cerevisiae IF01953, CG1945, and HF7C. Also, you may construct a new flocculant yeast. For example, the following Example 1 According to the method of MD Rose et al. (Methods in Yeast Genetics, 1990, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), a coagulation yeast ATCC60712 and a non-aggregation yeast W303-1B were used for conjugation. From the diploid, the cohesive yeast YF207 and yeast having properties equivalent thereto can be obtained. The flocculent yeast strain YF207 obtained by the present inventors has excellent plasmid stability and extremely high fermentation ability. Therefore, when the flocculent yeast strain YF207 which has been recombined so as to express dalcoamylase on the cell surface is used, the productivity of ethanol becomes extremely high.

The yeast displaying the dalcoamylase used in the method of the present invention on the cell surface can be obtained by introducing the above DNA into the yeast. Introduction of DNA means that DNA is introduced into yeast and expressed. Methods for introducing DNA include methods such as transformation, transduction, transfection, cotransfection, and electoral poration.Specific examples include the method using lithium acetate and the protoplast method. .

The introduced DNA may be integrated into the chromosome in the form of a plasmid, inserted into a host gene, or undergoing homologous recombination with the host gene.

The yeast into which DNA has been introduced is selected with a selectable marker (for example, TRP), and is selected by measuring glucose amylase activity. The immobilization of darcoamylase on the cell surface can be confirmed by an immunoantibody method using an anti-darcoamylase antibody and a FITC-labeled antibody.

The yeast that presents dalcoamylase to the cell surface used in the method of the present invention may contain DNA encoded to secrete dalcoamylase. In addition, the enzyme presenting dalcoamylase to the cell surface used in the method of the present invention was coded to express amylase, an endo-type hydrolase that solubilizes starch, on the cell surface or secrete it extracellularly. Including DN Α You can stay.

The yeast which displays the dalcoamylase used in the method of the present invention on the cell surface may be immobilized on a carrier. When fixed, it is convenient for use in repeated batch or continuous fermentations.

In the present specification, the carrier means a substance capable of immobilizing yeast, and is preferably a substance that is insoluble in water or a specific solvent. As the material of the carrier that can be used in the present invention, for example, foams or resins such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, silicon foam, and cellulose porous body are preferable. A porous carrier is preferred in consideration of the growth of yeast and the elimination of yeast that has decreased or has lost activity. The size of the opening of the porous material varies depending on the cell, but it is appropriate that the yeast can sufficiently enter and grow, and the size is preferably 50 πι to 1,000 μιη, but is not limited to this. Not done.

The shape of the carrier is not limited. Taking into account the strength of the carrier, culture efficiency, etc., it is spherical or cubic. The size is 2 mm to 50 mm in diameter for a sphere, and 2 mn! For a cubic. 55 O mm square is preferred.

In the present specification, the immobilization of yeast means a state in which the yeast is not in a free state, for example, a state in which the yeast is bound to or adhered to a carrier or incorporated into the carrier. For the immobilization of yeast, for example, a method commonly used by those skilled in the art such as a carrier binding method, a cross-linking method, and a comprehensive method can be applied. Above all, the carrier binding method is most suitable for immobilizing cohesive yeast. The carrier binding method includes a chemical adsorption method or a physical adsorption method in which the resin is adsorbed on an ion-exchange resin.

The aggregating yeast used in the method of the present invention, which displays dalcoamylase on the cell surface, has the property of being able to proliferate despite being immobilized on a carrier, and of being naturally dropped off when the activity is reduced. To bind to the carrier The characteristic of yeast is that the viable cell count is kept almost constant and the activity is high. Considering this feature, physical adsorption is most preferred for binding to the carrier. No special measures are required for physical adsorption. By simply mixing and culturing the cohesive or adhesive cells and the porous carrier, the cells enter the openings of the porous body and adhere to the carrier.

In the present specification, cohesiveness means a property in which yeasts or the like suspended or dispersed in a liquid are aggregated to form a lump (aggregate). Or, it means the property of combining to form an aggregate.

As used herein, the term "reduced activity" refers to a state in which the yeast itself is not killed, but the activity of the whole cell is weakened, or, for example, a DNA encoding an enzyme related to aggregation that has reduced activity related to aggregation. It is a state where the activity is weakened at the level and it becomes impossible to aggregate.

Further, in the present invention, the agglutinating or adhesive yeast may be a yeast to which the aggregating or adhesive property has been imparted by introducing a gene relating to agglutination or adhesion.

The gene relating to aggregation or adhesion includes a substance involved in aggregation or adhesion, for example, a structural gene encoding chitin, lectin, etc. in yeast, and the gene relating to aggregation is FL〇1 (J. Watari et al.). Agric. Biol. Chem., 55: 1547 (1991), GG Stewart et al., Can.J. Microbiol., 23: 441 (1977), I. Russell et al., Inst. Brew., 86: 120 (1980). , CW Lewis et al .: Γ · Inst.

Brew., 82: 158 (1976)), FLO 5 (I. Russell et al., J. Inst. Brew., 85: 95 (1979)), and FL08 (I. Yamashita et al., Agric. Biol. Chem., 48). : 131 (1 984)).

These genes related to aggregation or adhesion are incorporated into the above-described starting material plasmid and introduced into yeast along with DNA designed to display dalcoamylase on the cell surface. The immobilized yeast thus obtained can be cultured in a floating state while attached to a carrier, or packed in a column or the like, and used as a so-called bioreactor. Even when cultivation and fermentation are performed continuously or in batches (batch), the cells with reduced or dead activity are detached, so that the yeast activity is not reduced and the yeast can be used effectively. it can.

A method for producing ethanol by fermenting yeast presenting dalcoamylase on the cell surface according to the present invention in the presence of starch will be described.

The yeast that presents dalcoamylase on the cell surface 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 grow using starch as a carbon source, and when the soluble ^ fe starch is used, the starch concentration in the medium during culture is preferably a limit concentration of soluble starch. It is about 10 g / l, more preferably about 2 to about 6 g / l, most preferably about 4 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. The concentration of dissolved oxygen in the medium during aerobic cultivation is preferably about 0.5 to about 6 ppra, more preferably about 1 to about 4 ppm, and most preferably about 2.0 ppm. The temperature during the culture is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C. The cultivation time is preferably until the cell concentration reaches 10 g / l or more, and is about 20 to about 50 hours.

Next, the yeast displaying dalcoamylase on the cell surface is fermented under anaerobic conditions to produce ethanol. Examples of the form of the fermentation process include a batch (batch) process, a fed-batch batch process, a repetitive batch process, and a continuous process. Preferably, it is a repeated batch process or a continuous process.

Batch fermentation is the process of inoculating a yeast medium into a fermenter. This is a closed fermentation method performed by The time of the batch fermentation process can be determined according to the target alcohol concentration.

In the fed-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 supply of starch in each batch is preferably such that the initial starch concentration in the fermentor is from about 40 to about 150 g / l, more preferably from about 60 to about 120 g / l. The time for performing the fed-batch process may be determined according to the target alcohol concentration.

The repeating 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 the yeast, extracting the culture medium, and then adding a fresh medium to perform the fermentation step is repeatedly performed. The time of one batch process may be determined according to the target alcohol concentration.

A continuous fermentation process is a process in which fresh medium is continuously supplied to the fermenter while simultaneously removing the medium containing the product (ie, ethanol) from the fermenter. In the continuous fermentation process, the operation is performed with the supply rate of fresh medium equal to the discharge rate of medium containing ethanol. The feed rate of the culture medium is preferably about 0.01 to about 0.4 v / v% Z hours, more preferably about 0.07 to about 0.2 v / v% / hour of the amount of the culture medium in the fermenter. . In the continuous fermentation step, the yeast is preferably fixed to a carrier in the fermenter.

The concentration of starch added to the medium during fermentation is preferably about 40 to about 150 g / l. In particular, in the case of a repeated batch process, 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. The pH of the medium during fermentation is preferably from about 4.0 to about 6.0, most preferably about 5.0.

The concentration of dissolved oxygen in the medium during anaerobic fermentation is preferably about 1.Ορρπι or less, Preferably about 0. LPPM less, and most preferably about 0. 05p _P m. The temperature during fermentation is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C.

Since the above fermentation conditions change as the fermentation progresses, it is preferable to adjust these to a certain range. Changes over time in fermentation may be monitored by means commonly used by those skilled in the art, such as, for example, gas chromatography and HPLC.

After completion of the fermentation step, a medium containing ethanol is withdrawn from the fermenter, and ethanol is isolated by a separation process commonly used by those skilled in the art, for example, a separation operation using a centrifuge 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 1: Preparation of yeast displaying dalcoamylase on cell surface)

Saccharomyces diastaticus ATCC60712 (MATa leu2-3, 1 12 his2 lys2 stal FL08), which is an agglutinating yeast, and W303-IB (ΜΑΤ α ura3-52 trpl A 2 leu2- 3, 112 his3-, which is a non-aggregating yeast 11 ade2-1 cant 100) and according to the method of MD Rose et al. (Supra), a new agglutinating strain of tributofan auxotrophy YF207 (MATa ura3-52 trpl A 2 his ade2-1 canl) -100 stal FL08).

Multicopy plasmid pGA11 and chromosomal integration plasmid p

1 GA 1 1 (all provided by Tanaka Lab., Department of Synthesis and Biochemistry, Graduate School of Engineering, Kyoto University; Fig. 1) was converted to acetic acid using Yeast Maker (Clontech Laboratories, Inc., Palo Alto, CA). Yeast YF by lithium method

Introduced in 2007. The chromosome-integrated plasmid IGA11 was digested with the restriction enzyme Apal and then introduced into yeast. This is used as a selective medium,

SD agar supplemented with appropriate amino acids and bases without L-tryptophan Medium (6.7 g / L yeast nitrogen base w / o amino acids (Difco Laboratories), 2% glucose, 0.02 g / L adenine sulfate, 0.02 g / L L-histidine 'HC1, 0.03 g / L L-leucine, 0.02 g / L L-lysine and 0.02 g / L rasinole). The grown yeasts were selected and named YF207ZpGA11 and YF207 / pIGA11, respectively.

(Example 2: Confirmation of the function of yeast presenting dalcoamylase on the cell surface) The strains YF207 / pGA11 and YF207 / pIGA11 obtained in Example 1 were subjected to cell surface dalcoamylase. Was confirmed by measuring the gnorecoamylase activity as follows.

Soluble starch was added to a boiled sodium acetate buffer (pH 4.6) to a concentration of 0.5% to prepare a substrate solution. After holding 0.9 ml of the substrate solution at 30 ° C. for 5 minutes, 0.1 ml of the cell suspension was added, and the mixture was incubated at 30 ° C. for 15 minutes. The reaction was stopped by boiling for 10 minutes, and the concentration of the resulting glucose was measured using a commercially available kit, Glucose CII Test Co. (Wako Pure Chemical Industries, Ltd.), using a spectrophotometer (U-2001, manufactured by Hitachi, Ltd.). ) Was used to determine the absorbance at 505 mn. One unit of darcoamylase was defined as the amount of enzyme required to release 1 μιοΐ glucose per minute from starch. As a result, YF207 / pGAl1 and YF207 / pIGA11 express dalcoamylase of about 1 to 4 units Zg dry cells and about 0.5 to 2 units / g dry cells, respectively. I understand. The dry cell weight was measured as follows. A sample of 1 ml was taken into an eppendorf tube and pelleted by centrifugation at 6000 rpm for 5 minutes. After removing the supernatant, the pellet was resuspended in 1 ml of distilled water and again pelletized by centrifugation and dried. The weight of the tube containing the pellet was measured, and the dry cell weight was determined from the difference from the weight of the empty Etpendorf tube. In addition, the aggregation ability of the new strains YF207pGAl1 and YF207 / pIGA11 obtained in Example 1 was determined by the method of Smit et al. (Smit et al., Appl. Environ. Microbiol., 58: 3709-3714). (1992)). As a result, these strains exhibited the same strong agglutinating ability as YF207 before introducing the plasmid. This indicates that the expression of dalcoamylase on the cell surface does not affect the aggregation ability of yeast.

(Example 3: Cultivation of yeast displaying guar / recoamylase on cell surface)

Seed culture was performed by inoculating 5 ml of each yeast obtained in Example 1 into 100 ml of SD medium containing 1 ° / ₀ casamino acid (manufactured by Difco Laboratories) and shaking at 30 ° C for 48 hours. went.

Next, 50 ml of each seed culture was mixed with 1 L of 4% YPS medium (10 g / L yeast extract (manufactured by Dco Laboratories), 20 g / L polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 40 g / L starch (soluble ) (Made by Wako Pure Chemical Industries, Ltd.) and 5 g / L glucose) into a 2 L jar arm mentor (BMJ-02PI, Biott Corp., Tokyo), respectively, and aerobically at 30 ° C. Culture under specific conditions. The pH of the medium was maintained at 5.0 with sulfuric acid and sodium hydroxide, 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 strains thus cultured were used in the following various fermentation steps.

(Example 4: Production of ethanol by batch fermentation process using YF207 / pGAl1)

After performing the culture described in Example 3 for about 35 hours, the recovered cell pellet of YF207 / pGA11 was added to 1 L of 6% YPS medium (i.e., 60 g / L (Including starch) at pH 5.0, 30 ° C under anaerobic conditions The fermentation was performed for about 35 hours with gentle stirring (150 rpm). Starch, glucose, ethanol, dry cell weight, dalcoamylase activity, and plasmid stability were monitored throughout the culture and fermentation process. The glucose concentration was measured using a glucose _{CI I} Test Co., Ltd. (manufactured by Wako Pure Chemical Industries, Ltd.) and a spectrophotometer (U-2001, manufactured by Hitachi).

The starch concentration was measured as follows. That is, cells were separated from a 1.0 ml sample by centrifugation at 5 OOOrpm for 5 minutes, and the supernatant was diluted with distilled water and used for starch concentration measurement. A dalcoamylase solution from Aspergillus niger (6100 units / ml, Sigma Chemical Co., St. Louis, MO) was diluted 100-fold with distilled water, and 0.1 ml of the dalcoamylase solution was added to 0.9 ml of the diluted sample. And 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 in the same manner as in the above-mentioned measurement of glucose concentration, and converted to starch concentration.

The ethanol concentration was measured using a gas chromatograph (Model GC-8; manufactured by Shimadzu Corporation) equipped with a flame ionization detector. The measurement conditions were as follows: column, Unisole 300 0, (GL Science Inc.) packed in a 3.0 mm x 3. lm glass; column temperature, 210 ° C; injector / detector temperature, 270 ° C; carrier gas, nitrogen (flow rate: 25 ml / min).

In addition, the plasmid stability was measured as follows. Samples were diluted in tryptophan-free SD medium and plated on YPD plates and tryptophan-free SD plates (M.D. Rose et al., Supra). After 48 hours of incubation at 30 ° C, the number of colonies on both plates was counted. Plasmid stability (X) was determined by comparing the number of colonies on YPD plates (A) with the number of colonies on SD plates without tryptophan (B). That is, X (%) B X 100 / A was set.

The result is shown in figure 2. The growth rate of yeast in aerobic culture is high, The concentration rapidly decreased. In the fermentation process, the production of starch (〇) and the production of ethanol (▲) were started without delay (Fig. 2 (a)). Ethanol production was fast (0.71 g / hr / L), and its concentration reached 25 g / L after 30 hours of fermentation. Plasmid stability! "Raw decreased slightly during culture but was maintained during the fermentation process (data not shown). In addition, dalcoamylase activity (▽) on the cells was rather elevated during the fermentation process. Was maintained.

(Example 5: Production of ethanol by repeated batch fermentation process using YF207 / pGA11)

After performing the first fermentation step for about 35 hours in the same manner as the batch fermentation step described in Example 4, the yeast was separated by centrifugation at 5000 rpm for 10 minutes. Since YF207 / pGAl1 is an agglutinating yeast, it can be separated from the medium by sedimentation. However, centrifugation was performed to completely recover the cells. The collected cells were inoculated into 1 L of fresh 6% YPS medium, and the fermentation process was performed again. This operation was repeated seven times over about 300 hours.

The results are shown in Figure 3. The ethanol production rates for 1 to 7 batches calculated from the ethanol concentration (▲) during the fermentation process were 0.71, 0.67, 0.56, 0.59, 0.67, 0.62, and 0.60 g / hr / L, respectively. The yields of ethanol from starch in one to seven batches were 58, 46, 49, 50, 59, 51, and 57%, respectively. Thus, the ethanol production rate and ethanol yield were maintained for about 300 hours of seven repeated fermentation steps. In addition, the activity (V) and plasmid stability (♦) of dalcoamylase displayed on the cell surface were maintained at the same level during prolonged fermentation. Therefore, YF207ZpGAll has a very high level of stability in ethanol production by this strain, despite the introduction of a multicopy type plasmid. Turned out to be something.

(Example 6: Production of ethanol by fed-batch fermentation process using YF207 / pGA11).

After culturing for about 35 hours in the same manner as described in Example 3 and the dry cell weight reached about 15 g / L, 500 ml of concentrated medium 1 (lg / L yeast extratato, 1 g / L polypeptone, 105 g / L starch and 7.5 g / L glucose) were supplied to a fermenter, and fermentation was performed at 30 ° C at pH 5.0 under anaerobic conditions. After the consumption of starch by the yeast decreased, supplemented with 500 ml of concentrated medium 2 (2 g / L yeast extratato, lg / L polypeptone, 140 g / L starch, 10 g / L glucose). Fermentation continued at C. After the starch consumption by the yeast decreased again, 500 mL of the fermentation medium was removed from the fermenter, and 500 mL of the concentrated medium 2 was supplied to the fermenter to continue the fermentation. The same feeding procedure was repeated again.

The results are shown in FIG. The ethanol concentration (▲) in the fermentation medium reached 76. Og / L after about 140 hours of fermentation. The glucose concentration in the fermentation medium remained low during the fermentation. However, starch () accumulated during the fed-batch fermentation process. This is because darcoamylase presented on the cell surface is insoluble due to the presence of insoluble starch in the enriched medium supplied on the way.

'It was thought that it could not be sufficiently disassembled.

(Example 7: Production of ethanol by continuous fermentation process using YF207 / pGA11)

Batch fermentation was performed using YF207 / pGA11 under the same medium and culture conditions as in Example 4, and after about 30 hours from the start of fermentation, continuous fermentation was started by switching to continuous fermentation. A medium with a starch concentration of 200 g / 1 was intermittently supplied at a supply rate of 0.2 Vr. About 90 hours after switching A steady state was confirmed, and continuous operation was performed for about 400 hours. Table 1 shows the analysis results of the continuously extracted medium.

As can be seen from Table 1, it was revealed that the stability of the plasmid was maintained for a long time, and that alcohol was produced stably.

Industrial applicability

According to the present invention, ethanol can be efficiently produced directly from starch without using a process of saccharifying starch by using yeast that displays dalcoamylase on the cell surface. In particular, in the method of the present invention, it is possible to produce ethanol with higher efficiency by performing a repeated batch fermentation step or a continuous fermentation step using yeast that displays extremely stable dalcoamylase on the cell surface. Can be. Among them, a recombinant yeast derived from the YF207 strain has a very high ethanol fermentation ability, a high stability in the method of the present invention, and is very useful. Therefore, the method of the present invention is effective for industrial use in ethanol production.

Claims

The scope of the claims

1. A method for producing ethanol, comprising a step of fermenting in the presence of starch using a flocculent yeast having DNA that has been recombined so that dalcoamylase is displayed on the cell surface.

2. The method according to claim 1, wherein the step of fermenting is a step of repeated batch fermentation or a step of continuous fermentation.

3. The method according to claim 2, wherein the fermentation step is performed in a medium containing 40-300 gZl of starch at pH 4-6 at 20-45 ° C under anaerobic conditions.

4. The method according to claim 2, wherein in the continuous fermentation step, the supply rate of the medium is 0.07 to 0.2 v / v% / hour of the amount of the medium.

5. The method of claim 1, wherein the DNA is in the form of a plasmid, and the plasmid is a multicopy vector or a chromosomal integration vector.

6. The method according to claim 1, wherein the flocculating yeast is derived from strain YF207.

7. The method according to claim 8, wherein the flocculent yeast having the DNA is YF207 / pGA11 or YF207 / pIGALL.

8. A method for producing ethanol, comprising a step of repeatedly performing batch fermentation or continuous fermentation in the presence of starch using a non-aggregating yeast having a DNA that has been recombined so that dalcoamylase is displayed on the cell surface.

9. The method according to claim 8, wherein the fermentation step is performed in a medium containing 40-300 gZl of starch at pH 4-6 at 20-45 ° C under anaerobic conditions.

10. The method according to claim 8, wherein in the continuous fermentation step, the supply rate of the medium is from 0.07 to 0.2 vZv% Z hours of the amount of the medium.

11. The method according to claim 8, wherein the DNA is in the form of a plasmid, and the plasmid is a multicopy type vector or a chromosomal integration vector.

12. An aggregating yeast that displays dalcoamylase on the cell surface.