JP2012065604A - Recombinant yeast and method of producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which efficiently uses cellulose based biomass in a method of producing ethanol including a process which saccharifies cellulose based biomass using cellulase.SOLUTION: A recombinant yeast in which a β-glucosidase gene, a xylose reductase gene as a xylose metabolism related gene, and a β-xylosidase gene are introduced into the genome is disclosed, wherein the β-xylosidase gene is introduced in the form in which the expression control is carried out by the expression control region of a xylitol dehydrogenase gene, xylulokinase gene, and a hyperosmolarity response 7 (HOR7) gene. In addition, the method of producing ethanol includes a process which cultivates the recombinant yeast in a cellulose and/or hemicellulose containing medium including a cellulase formulation, and process which collects ethanol from the cellulose and/or hemicellulose containing medium.

Description

  The present invention relates to a recombinant yeast introduced with a predetermined gene and a method for producing ethanol using the recombinant yeast.

Cellulosic biomass is effectively used as a raw material for useful alcohols such as ethanol and organic acids. In order to improve ethanol production in ethanol production using cellulosic biomass, yeasts have been developed that can use xylose of 5 monosaccharides as a substrate (Patent Documents 1 and 2).
On the other hand, in ethanol production using cellulosic biomass, ethanol production efficiency can be improved by performing ethanol fermentation simultaneously with saccharification. For example, techniques for imparting saccharification-related enzymes to yeast responsible for ethanol fermentation are known (Patent Documents 3 and 4 and Non-Patent Document 1).

  In ethanol production using cellulosic biomass, in order to improve ethanol production and production efficiency, it is conceivable to add an enzyme preparation such as a cellulase preparation to a medium containing cellulosic biomass simultaneously with the use of yeast as described above. ing. However, when ethanol is produced from cellulosic biomass using the yeast described above, the amount of ethanol produced cannot be improved even when an enzyme preparation such as a cellulase preparation is added.

Special table 2000-509988 Special table 2008-506383 JP 2008-193935 A JP 2010-035556 A S. Katahira et al., Appl. Environ. Microbiol. Sept. 2004, p. 5407-5414

  Therefore, in view of the above-described circumstances, the present invention is a recombinant yeast that can efficiently use cellulosic biomass, particularly in a method for producing ethanol including a step of saccharifying cellulosic biomass using a cellulase preparation, and It aims at providing the manufacturing method of ethanol using the said recombinant yeast.

  As a result of intensive studies by the present inventors in order to achieve the above object, the xylosidase activity contained in the cellulase preparation is weak in the ethanol production method including the step of saccharifying cellulosic biomass using the cellulase preparation. It was clarified that saccharification did not proceed sufficiently due to this. In addition, the present inventors promoted saccharification of cellulosic biomass when a β-xylosidase gene is further introduced into the yeast as compared with a yeast introduced with a xylose metabolism-related gene and a β-glucosidase gene, The inventors have found that the productivity of ethanol is greatly improved and have completed the present invention.

  The present invention includes the following.

(1) A recombinant yeast in which a β-glucosidase gene, a xylose metabolism-related gene, and a β-xylosidase gene are introduced into the genome.
(2) The recombinant yeast according to (1), wherein the β-xylosidase gene is a β-xylosidase gene derived from Aspergillus oryzae.

(3) The recombinant yeast according to (1), wherein the β-glucosidase gene and the β-xylosidase gene are introduced as cell surface display type genes.
(4) The recombinant yeast according to (1), wherein the xylose metabolism-related genes are a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene.

(5) The recombinant yeast according to (1), wherein the gene is introduced using a higher-ploidy yeast as a host.
(6) The recombinant yeast according to (1), wherein the β-xylosidase gene is introduced in such a manner that its expression is controlled by the expression control region of the hyperosmotic response 7 (HOR7) gene.

(7) A step of culturing the recombinant yeast according to any one of (1) to (6) in a cellulose and / or hemicellulose-containing medium containing a cellulase preparation, and recovering ethanol from the cellulose and / or hemicellulose-containing medium. A process for producing ethanol comprising the steps.
(8) The method for producing ethanol according to (7), wherein the cellulose and / or hemicellulose-containing medium further contains cellulose derived from cellulosic biomass.

(9) The method for producing ethanol according to (7), wherein the culturing step is a step of saccharification of cellulose and / or hemicellulose and ethanol fermentation.
(10) The method for producing ethanol according to (7), wherein the culturing step is performed in an optimum temperature range for ethanol fermentation.

  The recombinant yeast according to the present invention has an activity to hydrolyze xylooligosaccharide to xylose and an activity to assimilate xylose to ethanol. For this reason, by utilizing the recombinant yeast according to the present invention, saccharification of cellulose and / or hemicellulose by a cellulase preparation can be promoted, and ethanol productivity can be greatly improved.

It is a figure which shows the process of producing pHPDC1-xylAfAG, pHHOR7-xylAfAG, and pHTDH2-xylAfAG. It is a characteristic view which shows the decomposition | disassembly characteristic of xylo-oligosaccharide about TX1 strain | stump | stock. It is a characteristic view which shows the decomposition characteristic of xylo-oligosaccharide about TX3410 strain. It is a characteristic view which shows the decomposition characteristic of xylo-oligosaccharide about TX3411 strain. It is a characteristic view which shows the decomposition characteristic of xylo-oligosaccharide about TX3412 strain | stump | stock. It is a characteristic view which shows ethanol productivity of TX1 strain, TX3410 strain, TX3411 strain and TX3412 strain. It is a characteristic view which shows the result of having measured PNPG activity about TX3411 strain, OC2-HUT strain, OC2-XR-XDH-HK strain, and TX1 strain. FIG. 3 is a characteristic diagram showing the results of culturing TX3411 strain, OC2-HUT strain, OC2-XR-XDH-HK strain, and TX1 strain and measuring ethanol production over time. FIG. 4 is a characteristic diagram showing the results of culturing TX3411 strain, OC2-HUT strain, OC2-XR-XDH-HK strain, and TX1 strain while changing the amount of enzyme used and the like, and measuring the amount of ethanol produced over time.

Hereinafter, the present invention will be described in more detail with reference to the drawings and examples.
<Recombinant yeast>
In the recombinant yeast according to the present invention, a β-glucosidase gene, a xylose metabolism-related gene, and a β-xylosidase gene are introduced into the genome. By expressing these β-glucosidase gene, xylose metabolism-related gene, and β-xylosidase gene, the recombinant yeast according to the present invention has β-glucosidase activity, xylose metabolism activity, and β-xylosidase activity.

  Here, β-glucosidase activity means an activity of catalyzing a reaction of hydrolyzing a β-glycoside bond of a sugar. That is, β-glucosidase can decompose cellooligosaccharides such as cellobiose into glucose. The xylose metabolic activity, as will be described in detail later, means the activity of assimilating xylose and producing ethanol using the metabolic pathway of ethanol fermentation inherently possessed by the yeast serving as the host. Furthermore, the β-xylosidase activity means an activity that catalyzes a reaction of hydrolyzing a xylo-oligosaccharide such as xylobiose into xylose.

  Of these β-glucosidase genes, xylose metabolism-related genes, and β-xylosidase genes, the β-glucosidase gene and the β-xylosidase gene are preferably introduced as cell surface display type genes, respectively. Here, the cell surface display type gene is a gene modified so that a protein encoded by the gene is expressed so as to be displayed on the surface layer of the cell. For example, the cell surface display type β-glucosidase gene is a gene obtained by fusing a β-glucosidase gene and a cell surface localized protein gene. The cell surface localized protein refers to a protein that is fixed on the cell surface layer of yeast and is present on the cell surface layer. For example, α- or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned. In general, a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal on the C-terminal side. Although it has the same secretory protein in that it has a secretion signal, the cell surface localized protein is different from the secreted protein in that it is transported by being immobilized on the cell membrane via a GPI anchor. When the cell surface localized protein passes through the cell membrane, the GPI anchor attachment recognition signal sequence is selectively cleaved, and is bound to the GPI anchor at the newly protruding C-terminal portion and fixed to the cell membrane. Thereafter, the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Next, the protein separated from the cell membrane is incorporated into the cell wall, fixed to the cell surface layer, and localized on the cell surface layer (see, for example, JP-A-2006-174767).

β-glucosidase gene The β-glucosidase gene is not particularly limited, and examples thereof include a β-glucosidase gene derived from Aspergillus aculeatus (Murai et al., Appl. Environ. Microbiol. 64: 4857-4861). In addition, as a β-glucosidase gene, a β-glucosidase gene derived from Aspergillus oryzae, a β-glucosidase gene derived from Clostridium cellulovorans, a β-glucosidase gene derived from Saccharomycopsis fibligera, and the like can be used.

Genes related to xylose metabolism Xylose metabolism-related genes are xylose reductase gene encoding xylose reductase that converts xylose to xylitol, xylitol dehydrogenase gene encoding xylitol dehydrogenase that converts xylitol to xylulose, and xylulose 5- It is meant to include a xylulokinase gene encoding xylulokinase that produces phosphate. Xylulose 5-phosphate produced by xylulokinase enters the pentose phosphate pathway and is metabolized.

  Examples of genes related to xylose metabolism introduced into the yeast genome include, but are not limited to, xylose reductase gene and xylitol dehydrogenase gene derived from Pichia stipitis, and xylulokinase gene derived from Saccharomyces cerevisiae (Eliasson A. et al. , Appl. Environ. Microbiol, 66: 3381-3386 and Toivari MN et al., Metab. Eng. 3: 236-249). In addition, a xylose reductase gene derived from Candida tropicalis or Candida prapsilosis can be used as the xylose reductase gene. As the xylitol dehydrogenase gene, a xylitol dehydrogenase gene derived from Candida tropicalis or Candida prapsilosis can be used. A xylulokinase gene derived from Pichia stipitis can also be used as the xylulokinase gene. A xylose isomerase gene derived from Streptomyces murinus cluster or the like can also be used.

β-xylosidase gene The β-xylosidase gene means a gene encoding a protein having an activity of catalyzing a reaction of hydrolyzing xylo-oligosaccharide to xylose. The β-xylosidase gene is not particularly limited, and any gene derived from any species can be used.

  In particular, as the β-xylosidase gene, an xylA gene derived from Aspergillus oryzae, an xlnD gene derived from Aspergillus nidulans, an xlnD gene derived from Aspergillus niger, a bxlI gene derived from Trichoderma reesei, and the like can be used.

  As an example, the base sequence of the coding region of the xylA gene derived from Aspergillus oryzae is shown in SEQ ID NO: 1, and the amino acid sequence of β-xylosidase encoded by the xylA gene is shown in SEQ ID NO: 2. In the present invention, the β-xylosidase gene is not limited to the one having the nucleotide sequence of SEQ ID NO: 1, but 80% or more similarity, preferably 90% or more similarity to the amino acid sequence of SEQ ID NO: 2. More preferably, a gene encoding a protein having an amino acid sequence having a similarity of 95% or more and having β-xylosidase activity may be used. Here, the similarity between base sequences can be calculated using homology search software such as FASTA and BLAST with default settings.

  Whether or not a given protein has β-xylosidase activity can be evaluated by adding the protein to a reaction solution containing a xylooligosaccharide such as xylobiose and measuring the amount of xylose produced.

Production of Recombinant Yeast The recombinant yeast according to the present invention can be produced by introducing the β-glucosidase gene, the xylose metabolism-related gene and the β-xylosidase gene described above into the yeast genome serving as a host.

  When introducing the above-described genes, it is preferable to introduce each gene in multiple copies. A method for introducing each gene in multiple copies is not particularly limited. For example, a method using homothallic properties of yeast having a plurality of selection markers can be applied. Japanese Patent Application Laid-Open No. 2009-34036 discloses a technique that makes it possible to easily introduce a multicopy gene into a genome by using yeast having homothallic properties. For example, by using the method disclosed in JP-A-2009-34036 and the yeast for transformation, each gene described above can be introduced in multiple copies.

  Yeast having homothallic properties is synonymous with homothallic yeast. The yeast having homothallic properties is not particularly limited, and any yeast can be used. Examples of yeast having homothallic properties include, but are not limited to, Saccharomyces cerevisiae OC-2 strain (NBRC2260). Other yeasts with homothallic properties include alcoholic yeasts (Taken 396, NBRC0216) (Source: “Characteristics of Alcoholic Yeast”, Sakekenkai Bulletin, No37, p18-22 (1998.8)), isolated in Brazil and Okinawa Ethanol producing yeast (Source: “Genetic properties of wild strains of Saccharomyces cerevisiae isolated in Brazil and Okinawa” Journal of Japanese Agricultural Chemical Society, Vol. 65, No. 4, p759-762 (1991.4)) and 180 (Source: Alcohol The screening of yeast having a strong fermenting ability ”, Journal of Japan Brewing Association, Vol.82, No.6, p439-443 (1987.6)). In addition, even a yeast exhibiting a heterothallic phenotype can be used as a yeast having homothallic properties by introducing the HO gene so that it can be expressed. That is, in the present invention, the yeast having homothallic properties is meant to include yeast into which the HO gene has been introduced so as to be expressed.

  Of these, the Saccharomyces cerevisiae OC-2 strain is preferable because it is a strain that has been confirmed to be safe and has been used in the winemaking scene. Further, the Saccharomyces cerevisiae OC-2 strain is preferable because it has excellent promoter activity under conditions of high sugar concentration, as shown in the Examples described later. In particular, the Saccharomyces cerevisiae OC-2 strain is preferable because it has an excellent promoter activity of the pyruvate decarboxylase gene (PDC1) under high sugar concentration conditions.

  Further, the promoter of the gene to be introduced is not particularly limited. For example, the promoter of glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), the promoter of 3-phosphoglycerate kinase gene (PGK1), the hyperosmotic response 7 gene ( HOR7) promoters can be used. Of these, the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene.

  That is, the gene described above may be introduced into the genome of yeast having homothallic properties together with a promoter controlling expression and other expression control regions. Alternatively, the above-described gene or its cell surface display-type gene may be introduced so that expression is controlled by a promoter of a gene originally present in the genome of yeast having homothallic properties or other expression control regions. In particular, it is preferable to introduce the gene described above downstream of the expression control region containing the promoter of the hyperosmotic response 7 (HOR7) gene in yeast having homothallic properties, that is, so that the expression is controlled by the expression control region. .

  In addition, as a method for introducing the above-described gene, any conventionally known technique known as a yeast transformation method can be applied. Specifically, for example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”, acetic acid Lithium method “J. Bacteriology, 153, p163 (1983)”, Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc. The method can be implemented, but is not limited thereto.

  The cell surface type β-glucosidase gene and the cell surface type β-xylosidase gene can also be introduced into the yeast genome by applying the materials and methods described above. Yeast into which a cell surface type gene has been introduced is a so-called arming yeast. Arming yeast is a new type that does not exist in normal yeast by fusing cell surface localized proteins with various functional proteins (enzymes, antigens, antibodies, reporter proteins, etc.) and peptides and displaying them on the cell surface. A yeast cell having a function or having an originally enhanced function. The cell surface layer is a periplasm which is a cell membrane, a cell wall, and a space between them, and a yeast cell that uses these layers and satisfies the above requirements is called an arming yeast. That is, a yeast that presents β-glucosidase and β-xylosidase on the cell surface type can be created based on published patent publications (JP-A-11-290078, WO02 / 085935) detailing the creation of arming yeast.

<Ethanol production>
Since the recombinant yeast described above has a β-xylosidase gene (preferably a cell surface display β-xylosidase gene) introduced therein, it can exhibit β-xylosidase activity. That is, the recombinant yeast can produce xylose by hydrolyzing xylooligosaccharides such as xylobiose contained in the medium. In addition, since the recombinant yeast has introduced a xylose metabolism-related gene, xylose can be assimilated to produce ethanol. Therefore, the recombinant yeast can produce xylose from the xylooligosaccharide contained in the medium, and can assimilate the xylose contained in the medium and the produced xylose to produce ethanol.

  Since the recombinant yeast has a xylose producing ability and an ethanol producing ability as described above, it is preferably used for so-called simultaneous saccharification and fermentation treatment using a medium containing cellulose and / or hemicellulose. Here, the simultaneous saccharification and fermentation treatment means a treatment that is carried out simultaneously without distinguishing between the step of saccharifying cellulosic biomass and the ethanol fermentation step. Prior to the simultaneous saccharification and fermentation treatment, a conventionally known pretreatment may be applied to the cellulosic biomass. Although it does not specifically limit as pre-processing, For example, the process which decomposes | disassembles a lignin with microorganisms, the grinding | pulverization process of a cellulose biomass, etc. can be mentioned.

  The saccharification method is not particularly limited, and examples thereof include an enzyme method using a cellulase preparation such as cellulase or hemicellulase. The cellulase preparation contains a plurality of enzymes involved in the degradation of cellulose chains and hemicellulose chains, and exhibits a plurality of activities such as endoglucanase activity, endoxylanase activity, cellobiohydrolase activity, glucosidase activity, and xylosidase activity. Although it does not specifically limit as a cellulase formulation, For example, the cellulase which Trichoderma reesei, Acremonium cellulolyticus, etc. produce can be mentioned. As the cellulase preparation, a commercially available product may be used.

  In the simultaneous saccharification and fermentation treatment, a cellulase preparation and the above-described recombinant microorganism are added to a medium containing cellulosic biomass (which may be after pretreatment), and the recombinant yeast is cultured in a predetermined temperature range. Although it does not specifically limit as culture | cultivation temperature, Considering the efficiency of ethanol fermentation, it can be set to 25-45 degreeC, and it is preferable to set it as 30-40 degreeC. Further, the pH of the culture solution is preferably 4-6. Moreover, you may stir and shake in the case of culture | cultivation.

  As shown in Examples described later, the cellulase preparation used for the simultaneous saccharification and fermentation treatment has a low β-xylosidase activity among a plurality of enzyme activities involved in the degradation of the cellulose chain and the hemicellulose chain. Since the optimal reaction temperature of β-xylosidase is about 70 ° C., it has been reported that the enzyme activity decreases to about 1/3 around the culture temperature. That is, conventionally, when simultaneous saccharification and fermentation treatment is performed using a cellulase preparation, hydrolysis of xylooligosaccharides such as cellobiose is insufficient, and xylan and hemicellulose cannot be sufficiently used as xylose for ethanol fermentation. However, since the above-described recombinant yeast is used in the present invention, the amount of saccharification by cellulase preparation, particularly xylose, can be improved. As a result, in the simultaneous saccharification and fermentation treatment using the above-described recombinant yeast, more xylose can be used and ethanol productivity is improved.

  In the method for producing ethanol using the recombinant yeast according to the present invention, ethanol is recovered from the medium after the simultaneous saccharification and fermentation treatment. The method for recovering ethanol is not particularly limited, and any conventionally known method can be applied. For example, after the above-described ethanol fermentation is completed, a liquid layer containing ethanol and a solid layer containing recombinant yeast and solid components are separated by solid-liquid separation operation. Thereafter, ethanol contained in the liquid layer is separated and purified by a distillation method, whereby high purity ethanol can be recovered. The purity of ethanol can be adjusted as appropriate according to the intended use of ethanol.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1]
In this example, an OC2-HUT strain in which histidine requirement, uracil requirement and tryptophan requirement were imparted to the Saccharomyces cerevisiae OC-2 strain was used. The method for producing this OC2-HUT strain is described in detail in JP 2010-035556 A. In this example, 2 copies of the cell surface display β-glucosidase gene, 2 copies of the xylose metabolism-related gene, and 2 copies of the cell surface display β-xylosidase gene were introduced into the OC2-HUT strain.

<Plasmid preparation>
When introducing the cell surface display β-glucosidase gene, a plasmid in which pABGL-PDC1P (HIS3 marker) was linearized with the restriction enzyme AscI was used. The plasmid pABGL-PDC1P is described in detail in Japanese Patent Application Laid-Open No. 2010-035556.

  When introducing a gene related to xylose metabolism, a plasmid in which pIUX1X2Xk (URA3 marker) was linearized with the restriction enzyme NcoI was used. The plasmid pIUX1X2Xk is disclosed in S. Katahira et al., Appl. Microbiol. Biotechnol. (2006) 72: 1136-1143, and the xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene are respectively controlled by the GAPDH promoter. A plasmid introduced so as to allow expression below.

  Moreover, in this example, three types of plasmids (pHPDC1-xylAfAG, pHHOR7-xylAfAG and pHTDH2-xylAfAG) having different promoters used for introducing the cell surface-presenting β-xylosidase gene were prepared. The procedure for preparing these plasmids is shown in FIG.

  First, by pCUSxylAf disclosed in S. Katahira et al., Appl. Environ. Microbiol. Sept. 2004, p. 5407-5414 as a template, PCR using a pair of primers BssHII-EcoRI-NotI_GLAss_fw and BssHII_3′AGterm_rv A 3893 bp DNA fragment containing a signal peptide (“ss” in the figure), a β-xylosidase gene (XylA) derived from Aspergillus oryzae, a FLAG tag, and 3′-half α-agglutinin in this order was amplified. The obtained DNA fragment and pRS403 manufactured by Stratagene were each treated with BssHII, and then ligated to prepare plasmid pRS403-xylAfAG.

  On the other hand, a DNA fragment of about 650 bp containing the PDC1 promoter was amplified by PCR using the genomic DNA of S. cerevisiae YPH449 strain as a template and a pair of primers EcoRI_PDC1_fw and NotI_PDC1_rv. Similarly, an approximately 1000 bp DNA fragment containing the HOR7 promoter was amplified by PCR using a pair of primers EcoRI_HOR7_fw and NotI_HOR7_rv. Similarly, an about 1000 bp DNA fragment containing the HOR7 promoter was amplified by PCR using a pair of primers EcoRI_TDH2_fw and NotI_TDH2_rv.

Three types of DNA fragments containing these promoters were treated with EcoRI and NotI, respectively. The obtained three types of DNA fragments were ligated to plasmids pRS403-xylAfAG treated with EcoRI and NotI, respectively, to prepare three types of plasmids pHPDC1-xylAfAG, pHHOR7-xylAfAG, and pHTDH2-xylAfAG.
In addition, the base sequence of the primer used in the present Example is shown below.

<Transformation>
First, the plasmid pABGL-PDC1P treated with AscI was transformed into the OC2-HUT strain and selected with SD + uracil + histidine plate. Since the resulting transformant had a plasmid fragment introduced on one side of the chromosome, a tryptophan prototrophic strain was isolated after sporulation to produce a recombinant strain into which two copies of pABGL-PDC1P had been introduced.

  Next, plasmid pIUX1X2Xk treated with NcoI was transformed into the above recombinant strain. Frozen-EZ Yeast Transformation II (Zymo Research) was used for transformation. The selection plate was an SD plate, and colonies obtained by transformation were selected on an SD + histidine plate. Similarly, after spore formation, a uracil prototrophic strain was isolated to prepare a recombinant strain in which two copies of pIUX1X2XK were introduced into the above recombinant strain.

  Finally, pHCDC1-xylAfAG, pHHOR7-xylAfAG or pHTDH2-xylAfAG linearized with NdeI was transformed into the above strain. And the histidine prototrophic strain was acquired similarly. In this way, 2 copies of pABGL-PDC1P and 2 copies of pIUX1X2XK were introduced using the OC2-HUT strain as a host, and TX3410 strain into which 2 copies of pHPDC1-xylAfAG were introduced, TX3411 strain into which 2 copies of pHHOR7-xylAfAG were introduced, and A TX3412 strain into which two copies of pHTDH2-xylAfAG were introduced was prepared.

[Example 2]
In this example, the degradation characteristics of xylooligosaccharides were evaluated for the TX3410 strain, TX3411 strain and TX3412 strain prepared in Example 1 and the TX1 strain for comparison.

  The test was conducted as follows. In a culture solution containing 20% (w / w) xylooligosaccharide 95p (manufactured by Suntory), 1% (w / w) yeast extract and 2% (w / w) bacto peptone, the cell concentration is OD660 = Each strain was inoculated to be 4. The culture solution was 60 ml, and a 100 ml flask container was used. The culture temperature was 37 ° C., and the culture was performed while shaking at 150 rpm.

  At the start of the reaction, the concentrations of ethanol, xylose, xylobiose and xylotriose contained in the culture solution 6 hours, 24 hours, 30 hours and 48 hours after the start of the reaction were measured.

  The result of the test using TX1 strain for comparison is shown in FIG. As can be seen from FIG. 2, the xylose contained in the medium was completely consumed in about 24 hours from the start of the culture, and the amount of ethanol produced did not increase. This indicates that xylobiose and xylotriose cannot be decomposed into xylose because TX1 strain does not have β-xylosidase activity.

  Moreover, the result of the test using TX3410 strain, TX3411 strain and TX3412 strain is shown in FIGS. 3, 4 and 5, respectively. As shown in FIGS. 3 to 5, it can be seen that xylobiose and xylotriose in the medium decreased and xylose increased even after 48 hours from the start of culture. This indicates that the cell surface display β-xylosidase gene introduced into the TX3410 strain, TX3411 strain, and TX3412 strain functions, and that the TX3410 strain, TX3411 strain, and TX3412 strain have β-xylosidase activity. Further, as can be seen from FIGS. 3 to 5, when the TX3410 strain, TX3411 strain, and TX3412 strain are used, the production amount of ethanol is greatly improved as compared with the case where the TX1 strain is used. This indicates that the xylose metabolism-related genes introduced into the TX3410 strain, TX3411 strain and TX3412 strain are functioning, and the xylose produced by the β-xylosidase activity described above is used for ethanol fermentation.

  Furthermore, when the results shown in FIGS. 3 to 5 are compared, it can be seen that the ethanol production is most excellent when the TX3411 strain shown in FIG. 5 is used. That is, it is preferable to use the HOR7 promoter as the promoter that controls the expression of the cell surface-presenting β-xylosidase gene. By using the HOR7 promoter, the cell surface display-type β-xylosidase gene can be expressed at a higher level, and the ethanol productivity can be improved. From the results shown in FIGS. 2 to 5, FIG. 6 shows the results of a comparison of ethanol productivity of the TX1 strain, TX3410 strain, TX3411 strain and TX3412 strain collectively.

Example 3
In this example, TX3411 strain was used as a representative example, and PNPG activity in a model system for simultaneous saccharification and fermentation of cellulosic biomass was examined. The PNPG activity is an index of β-glucosidase activity. That is, since β-glucosidase generates p-nitrophenol and D-glucose using PNPG as a substrate, β-glucosidase activity can be verified by measuring the amount of p-nitrophenol. For PNPG activity, the amount of enzyme that produces 1 micromole of p-nitrophenol per minute under the following conditions was defined as 1 U.

The reagents used for the measurement are as follows.
・ 0.1M acetate buffer pH5.0 (25 ℃)
・ 20mM PNPG aqueous solution (603mg PNPG dissolved in 100ml distilled water)
・ 0.2M Na ₂ CO ₃ solution (21.2 g of anhydrous sodium carbonate dissolved in 1 liter of distilled water)
The measurement procedure was as follows. First, a reaction mixture (1.0 ml of 0.1 M acetate buffer and 0.5 ml of PNPG aqueous solution) was prepared in a test tube, and pre-warmed at 37 ° C. for about 5 minutes. Next, 0.5 ml of the diluted culture solution was added to start the reaction. Then, after exactly allowed to react for 15 minutes at 37 ° C., was added a solution of Na ₂ CO ₃ 2 ml, the reaction was stopped. Next, the 400 nm absorbance in the reaction solution was measured. In the blind test, the reaction solution was allowed to stand at 37 ° C. for 15 minutes, ₂ ml of Na ₂ CO ₃ solution was added and mixed, and then 0.5 ml of enzyme solution was added and adjusted.

  In this example, for comparison with the TX3411 strain, OC2-HUT strain, OC2-XR-XDH-HK strain in which only the plasmid pIUX1X2Xk was introduced into the OC2-HUT strain, and TX1 strain were similarly measured for PNPG activity. . In this example, medium composition: 4% (w / w) KC floc, 4% (w / w) Birchwood xylan, 1% (w / w) yeast extract, 2% (w / w) bacto Medium of peptone (w / w) and 3% (w / w) cellulase preparation (NS50013) was used. Each strain was inoculated so that the bacterial cell concentration was OD660 = 6. The culture solution was 30 ml, and a 100 ml flask container was used. The culture temperature was 37 ° C., and the culture was performed while shaking at 80 rpm.

  At the start of the reaction, PNPG activity was measured for the culture broth 6 hours, 24 hours, 30 hours and 48 hours after the start of the reaction. The result is shown in FIG. As can be seen from FIG. 7, the TX1 strain and TX3411 strain into which the cell surface-presenting β-glucosidase gene was introduced have higher PNPG activity over time from the start of the culture. In contrast, only a slight PNPG activity was detected in the OC2-HUT strain and the OC2-XR-XDH-HK strain that do not have the β-glucosidase gene. The PNPG activity detected when these OC2-HUT strain and OC2-XR-XDH-HK strain were cultured is considered to be due to the cellulase preparation added to the medium. Although the TX3411 strain is lower than the PNPG activity of the TX1 strain, it has been shown to have an excellent β-glucosidase activity, having about four times the PNPG activity compared to the β-glucosidase activity of the cellulase preparation. . Therefore, it was shown that not only TX3411 strain but also TX3410 strain and TX3412 strain prepared in Example 1 have characteristics suitable for simultaneous saccharification and fermentation.

Example 4
In this example, the TX3411 strain was used as a representative example, and ethanol production in a model system for simultaneous saccharification and fermentation of cellulosic biomass was examined. In this example, the TX3411 strain, the OC2-HUT strain, the OC2-XR-XDH-HK strain, and the TX1 strain were cultured under the conditions described in Example 4, and the ethanol production was measured over time. The results are shown in FIG.

  As can be seen from FIG. 8, the TX3411 strain was able to achieve about 20% higher ethanol productivity than the TX1 strain even in the model system for simultaneous saccharification and fermentation of cellulosic biomass. That is, it has been clarified that the ethanol productivity can be improved in the actual simultaneous saccharification and fermentation treatment by introducing the cell surface-presenting β-xylosidase. When the OC2-HUT strain was used, ethanol could hardly be produced even though the culture solution contained a cellulase preparation. This is presumably because the cellulase activity of the cellulase preparation significantly decreases at a temperature (37 ° C.) far below the optimum temperature of 50 ° C. Therefore, it can be said that in the actual simultaneous saccharification and fermentation treatment, it is preferable to use a recombinant yeast having a xylose utilization property in which a β-xylosidase gene and a β-glucosidase gene are introduced in addition to the cellulase preparation.

  On the other hand, when OC2-XR-XDH-HK strain was used, 0.41% ethanol was produced 48 hours after the start of the culture. This indicates that the xylose metabolism-related gene introduced into the OC2-HUT strain has made ethanol production from xylose possible. When TX3411 strain was used, 0.75% ethanol was produced 48 hours after the start of culture. Compared to the case where only xylose metabolism related gene was introduced into OC2-HUT strain, cell surface display type By further introducing the β-xylosidase gene and the cell surface display β-glucosidase gene, the ethanol production was improved by 82%.

Example 5
In this example, TX3411 strain was used as a representative example, and the effect of reducing the amount of enzyme used in a model system for simultaneous saccharification and fermentation of cellulosic biomass was examined based on the amount of enzyme used and the amount of ethanol produced. In this example, for comparison, the relationship between the amount of enzyme used and the amount of ethanol produced was also calculated for the OC2-HUT strain, the OC2-XR-XDH-HK strain, and the TX1 strain.

  In this example, the TX3411 strain, the OC2-HUT strain, the OC2-XR-XDH-HK strain, and the TX1 strain were used under the medium and culture conditions described in Example 4 except that the enzyme usage was changed as shown in the following table. In addition, ethanol production was measured over time.

  FIG. 9 shows the results of measuring ethanol production over time for the test groups 1 to 7 shown in Table 2. Table 3 shows the results of measuring the amount of ethanol produced after 48 hours from the start of culture and calculating the amount of enzyme used per unit produced ethanol.

  As can be seen from FIG. 9 and Table 3, it was found that when the TX3411 strain was used (test groups 5 to 7), the amount of enzyme used could be reduced compared to other test groups. In particular, compared with the case of using OC2-XR-XDH-HK strain (test group 3), it can be seen that in test group 6, the amount of enzyme used can be reduced by about 60% or more. Not only the TX3411 strain shown in this example but also the TX3410 strain and TX3412 strain prepared in Example 1 can reduce the amount of enzyme during simultaneous saccharification and fermentation, and can be used for simultaneous saccharification and fermentation treatment at low cost. Indicated.

Claims (10)

  A recombinant yeast in which a β-glucosidase gene, a xylose metabolism-related gene and a β-xylosidase gene are introduced into the genome.   The recombinant yeast according to claim 1, wherein the β-xylosidase gene is a β-xylosidase gene derived from Aspergillus oryzae.   The recombinant yeast according to claim 1, wherein the β-glucosidase gene and the β-xylosidase gene are introduced as cell surface display type genes.   The recombinant yeast according to claim 1, wherein the xylose metabolism-related genes are a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene.   2. The recombinant yeast according to claim 1, wherein the gene is introduced using a higher-ploidy yeast as a host.   The recombinant yeast according to claim 1, wherein the β-xylosidase gene is introduced in such a manner that its expression is controlled by the expression control region of the hyperosmotic response 7 (HOR7) gene.   A step of culturing the recombinant yeast according to any one of claims 1 to 6 in a cellulose and / or hemicellulose-containing medium containing a cellulase preparation, and a step of recovering ethanol from the cellulose and / or hemicellulose-containing medium. A method for producing ethanol.   The method for producing ethanol according to claim 7, wherein the cellulose and / or hemicellulose-containing medium further contains cellulose derived from cellulosic biomass.   8. The method for producing ethanol according to claim 7, wherein the culturing step is a step of saccharification of cellulose and / or hemicellulose and ethanol fermentation.   The method for producing ethanol according to claim 7, wherein the culturing step is carried out in an optimum temperature range for ethanol fermentation.

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