JP2008086310A - Yeast performing surface display of cellulase and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a yeast enabling ethanol fermentation by easily and efficiently decomposing a polymer formed by bonding glucose through β-glucoside bond, and a method for easily and efficiently producing ethanol from a polymer formed by bonding the glucose through the β-glucoside bond. <P>SOLUTION: The polymer formed by the β-glucoside bonding of the glucose can be decomposed in high efficiency to enable ethanol fermentation by the use of a sake yeast in which both of β-glucosidase and endoglucanase are bonded to the cell membrane through a cell cortex localized protein. The enzymatic activity is remarkably improved by interposing a linker peptide having 5-10 amino acid residues between the β-glucosidase and the cell cortex localized protein and interposing a linker peptide having 14-23 amino acid residues between the endoglucanase and the cell cortex localized protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to sake yeast that co-presents β-glucosidase and endoglucanase on the surface, and a method for producing ethanol from plant raw materials using this yeast.

  In recent years, bioethanol produced from plant materials has attracted attention as a fuel source to replace fossil fuels. Bioethanol is useful in that it does not increase carbon dioxide on the earth even if it is burned because carbon dioxide in the air is derived from immobilized plant material. For example, ethanol blended gasoline is used in the United States and the like, and is scheduled to be used in Japan.

  Bioethanol is produced from saccharide raw materials such as sugar cane and sugar beet and starchy raw materials such as corn and wheat through microbial fermentation, which are also edible resources. In recent years when global food shortages are a concern, ethanol production technology from non-edible carbon sources such as waste materials and food residues is required and developed.

    Although a method has been devised that treats biomass containing cellulose and hemicellulose with acid treatment or supercritical treatment, and then treats the raw material to glucose that can be assimilated by fermentation microorganisms, these require strong acids or high temperatures and pressures. For example, overloads on facilities and high energy costs are cited as problems.

    On the other hand, research aimed at direct ethanol fermentation from non-food carbon sources has been performed on functional microorganisms using molecular biological techniques for fermenting microorganisms that cannot inherently assimilate cellulose or hemicellulose. We have not yet left the laboratory level in the laboratory.

  Here, “yeast cell surface display engineering” in which target proteins such as enzymes are displayed on the surface of yeast cells has attracted attention. This is a technique for presenting a target protein at a high density on the cell surface of yeast by expressing the target protein as a fusion protein with a protein that can be immobilized on the cell surface, such as a GPI anchoring protein.

  Since this cell surface display yeast has the following advantages, it is suitable for substance production using cells that produce enzymes.

  First, in this yeast, the enzyme, which is the target protein, is present on the cell surface, so that the permeation of the substrate through the cell membrane is not a problem, and cells are not easily damaged by the enzyme.

  Secondly, since this yeast can immobilize the enzyme in the cells at high density, the enzyme concentration in the reaction system is increased by filling or suspending the cells in the reaction tank at high density. As a result, it is relatively easy to scale up. In addition, since the enzyme is fixed outside the microbial cell, it is difficult to be affected by the proteolytic enzyme present in the microbial cell. In addition, it is expected that the action of a proteolytic enzyme existing outside the cell body is less likely to be affected due to the presence of the enzyme at a high density on the cell surface.

  Thirdly, a large amount of target enzyme can be easily obtained by growing this yeast, and the enzyme can be recovered and concentrated together with the cells very easily by centrifugation.

  Such “yeast cell surface display engineering” is a new research field, and various studies are currently being conducted at the laboratory level. For example, in Patent Document 1 and Patent Document 2, a polynucleotide comprising a highly active promoter, a secretory signal sequence that functions in yeast cells, a sequence encoding arabinofuranosidase, and a GPI anchoring protein is incorporated into the chromosome. A method for degrading xylan using yeast is described. By using yeast displaying surface layer of arabinofuranosidase and xylosidase or xylanase, xylan (hemicellulose) in plant material can be easily decomposed to obtain monosaccharide, and further, xylose reductase, xylitol dehydrogenase, and If xylulokinase, or xylose isomerase and xylulokinase are highly expressed and imparted xylose utilization ability, alcoholic fermentation can be performed.

However, the arabinofuranosidase-presenting yeast disclosed in Patent Documents 1 and 2 cannot alone decompose xylan into monosaccharides.
JP 2005-245334 A JP 2005-245335 A

    The present invention provides a yeast that can be efficiently and easily decomposed by ethanol fermentation using a β-glucoside bond of glucose, and a polymer of β-glucoside bond of glucose. It is an object of the present invention to provide a method capable of easily producing ethanol.

In order to solve the above-mentioned problems, the present inventor repeated research and obtained the following knowledge.
(i) Sake yeast having the following polynucleotides (a) and (b) incorporated in the chromosome co-presents β-glucosidase and endoglucanase on the surface. If this yeast is reacted with a polymer by β-glucoside bond of glucose in plant material, the above-mentioned rate of degradation of the polymer per weight of the cell and ethanol can be obtained rather than using two types of yeasts that display each enzyme on the surface. The generation speed is increased.
(a) a polynucleotide comprising a secretory signal sequence that functions in yeast cells, a sequence that encodes β-glucosidase, a cell surface localized protein, or a sequence that encodes a cell membrane binding region in this order from the 5 ′ end
(b) a polynucleotide comprising a secretory signal sequence that functions in yeast cells, a sequence encoding endoglucanase, a cell surface localized protein or a sequence encoding a cell membrane binding region in this order from the 5 ′ end side
(ii) A sequence encoding the endoglucanase and a cell surface layer by interposing a linker of 6 to 90 base length between a sequence encoding β-glucosidase and a sequence encoding a cell surface localized protein or a cell membrane binding region thereof. By interposing a linker having a length of 6 to 90 bases between the localized protein or the sequence encoding the cell membrane binding region, the polymer degradation rate per cell weight is remarkably increased.

  The present invention has been completed based on the above findings, and provides the following sake yeast and a method for producing ethanol.

Item 1. A sake yeast in which the following polynucleotides (a) and (b) are integrated into a chromosome, and β-glucosidase and endoglucanase are co-presented on the surface.
(a) a polynucleotide comprising, in order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes β-glucosidase, and a sequence that encodes a cell surface localized protein or a cell membrane binding region thereof
(b) a polynucleotide comprising, in order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes endoglucanase, and a sequence that encodes a cell surface localized protein or a cell membrane-binding region thereof. The polynucleotide of (a) is a linker having a length of 6 to 90 bases between a sequence encoding β-glucosidase and a sequence encoding a cell surface localized protein or a cell membrane binding region thereof,
The polynucleotide of (b) is a linker having a length of 6 to 90 bases between the sequence encoding endoglucanase and the sequence encoding the cell surface localized protein or its cell membrane binding region,
Item 2. The sake yeast according to item 1.

  Item 3. Item 3. The sake yeast according to item 1 or 2, wherein the secretion signal sequence of (a) is a combination of two or more secretion signal sequences.

  Item 4. Item 4. The sake yeast according to any one of Items 1 to 3, wherein the secretion signal sequence of (a) is a combination of two or more secretion signal sequences.

  Item 5. Item 5. The sake yeast according to any one of Items 1 to 4, wherein the secretory signal sequence in (a) is a combination of a secretory signal sequence originally possessed by secreted β-glucosidase and a different secretory signal sequence.

  Item 6. The secretory signal sequence of (a) is (1) the secretory signal sequence of Aspergillus oryzae-derived β-glucosidase (BGL7) and the secretory signal sequence of Aspergillus oryzae-derived β-glucosidase (BGL1) Or (2) the sake yeast according to any one of Items 1 to 5, wherein the secretory signal sequence of BGL7 and the secretory signal sequence of Rhizopus oryzae-derived glucoamylase (GlaR) are combined.

  Item 7. Item 7. The secretory signal sequence of (b) is a combination of the secretory signal sequence originally possessed by endoglucanase and the secretory signal of Aspergillus oryzae-derived endoglucanase (CelA). Sake yeast.

  Item 8. Item 8. The sake yeast according to any one of Items 1 to 7, wherein the secretory signal sequence of (b) is a combination of a secretory signal sequence of CelA and a secretory signal sequence of endoglucanase (CelB) derived from Aspergillus oryzae.

  Item 9. Item 9. The sake yeast according to any one of Items 1 to 8, wherein the nucleotide of (a) and / or (b) is incorporated into a multiple copy chromosome.

  Item 10. Item 10. The sake yeast according to any one of Items 1 to 9, wherein the sequence encoding the cell surface localized protein or the cell membrane binding region thereof is a sequence encoding 320 to 600 amino acids from the C-terminal of α-agglutinin of yeast.

  Item 11. Sake yeast in which β-glucosidase and endoglucanase are bound to a cell membrane via a cell surface localized protein or a cell membrane binding region thereof, respectively.

  Item 12. A linker peptide consisting of 2 to 30 amino acid residues is interposed between β-glucosidase and cell surface localized protein or its cell membrane binding region, and 2 between endoglucanase and cell surface localized protein or its cell membrane binding region. Item 12. The sake yeast according to Item 11, wherein a linker peptide consisting of ˜30 amino acid residues is interposed.

  Item 13. The manufacturing method of ethanol which decomposes | disassembles the polymer by the beta-glucoside bond of glucose in presence of the sake yeast in any one of claim | item 1-11, and obtains ethanol by fermentation of the produced | generated glucose.

  Item 14. Item 14. The method according to Item 13, wherein the polymer due to the β-glucoside bond of glucose is contained in a treated product obtained by hydrolyzing the plant material.

  Sake yeast in which the above polynucleotides (a) and (b) are incorporated into the chromosome, β-glucosidase and endoglucanase bind to the cell membrane via the cell surface localized protein or its cell membrane binding region, respectively. Are co-presented on the cell surface. If this yeast is used to decompose the polymer due to the β-glucoside bond of glucose, on the surface of the yeast, random decomposition of the polymer by endoglucanase and degradation by β-D-glucose unit by β-glucosidase, Since it is carried out while delivering the substrate in a narrow range near the cell surface, it is possible to efficiently decompose the polymer into D-glucose and further ferment it to obtain ethanol.

  In addition, a linker peptide of 2 to 30 amino acid residues is interposed between β-glucosidase and cell surface localized protein or its cell membrane binding region, and between endoglucanase and cell surface localized protein or its cell membrane binding region. When a linker peptide having 2 to 30 amino acid residues is interposed, the decomposition efficiency of the polymer and the ethanol production efficiency are remarkably improved.

  Further, in the sake yeast of the present invention, the β-glucosidase gene and endoglucanase gene are integrated into the chromosome, so that the expression unit is not easily dropped without applying selective pressure. For this reason, each enzyme can be highly expressed even in culture on an industrial scale where it is difficult to apply selective pressure in terms of cost.

  The sake yeast of the present invention can improve enzyme activity by optimizing the combination of secretory signal sequences.

  In the ethanol fermentation using the yeast of the present invention, when acid treatment or supercritical treatment is used in the pretreatment of the raw material biomass, it is not necessary to break down into glucose, so that it can be broken down under milder conditions. Therefore, it is possible to avoid overloading the equipment and high energy costs. In addition, if the raw material biomass is decomposed to glucose only by supercritical treatment, a hyperdegradable product is likely to be generated. However, if the raw material biomass is supercritically treated and then ethanol fermentation using the yeast of the present invention is performed, the superdegraded product Since the conditions under which the generation of odors is further suppressed can be adopted, an improvement in the raw material recovery rate can be expected.

  Furthermore, sake yeast can obtain a high growth rate even in culture on an industrial scale, which may result in severe culture conditions. Also, sake yeast has extremely high resistance to ethanol, so it can be used for ethanol fermentation as it is after producing D-glucose.

Hereinafter, the present invention will be described in detail.
(I) Sake Yeast The sake yeast of the present invention is a sake yeast in which the polynucleotides (a) and (b) described above are incorporated into a chromosome and β-glucosidase and endoglucanase are co-presented on the surface. In this sake yeast, β-glucosidase and endoglucanase are each bound to the cell membrane via a cell surface localized protein or a cell membrane binding region thereof.
Secretory signal sequence The secretory signal sequence is a polynucleotide sequence encoding a secretory signal peptide. A secretory signal peptide is a peptide that is normally bound to the N-terminus of a secretory protein that is secreted extracellularly, including the periplasm. This peptide has a similar structure between organisms, consists of about 20 amino acids, has a basic amino acid sequence in the vicinity of the N-terminus, and then contains many hydrophobic amino acids. The secretory signal is usually removed by degradation by a signal peptidase when the secreted protein is secreted from inside the cell through the cell membrane to the outside of the cell.

  In the present invention, any polynucleotide sequence encoding a secretory signal peptide capable of secreting β-glucosidase and endoglucanase out of the yeast cell can be used, regardless of its origin. . For example, secretion signal peptide sequence of glucoamylase such as Rhizopus oryzae, secretion signal peptide sequence of glucoamylase of Aspergillus oryzae, secretion signal of α- or a-agglutinin of yeast (Saccharomyces cerevisiae) Peptide sequences, secretory signal peptide sequences of α-factor derived from yeast (Saccharomyces cerevisiae), and the like can be suitably used. In particular, the secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase is preferable from the viewpoint of secretion efficiency. Also preferred is the secretory signal peptide sequence of Aspergillus oryzae-derived glucoamylase.

  The origin of the secretory signal sequence is not particularly limited, but it is preferably derived from a protein functionally or structurally similar to β-glucosidase and endoglucanase, and preferably derived from a sugar chain hydrolase. As long as the secretion signal sequence has the ability to secrete β-glucosidase or endoglucanase outside the yeast cell, any mutation such as amino acid substitution, addition, deletion, deletion, etc. is added to the known secretion signal sequence. It may be.

  More preferable secretion signal sequences include a secretion signal sequence of glucoamylase, a secretion signal sequence of β-glucosidase, a secretion signal sequence of endoglucanase, and a secretion signal sequence of exoglucanase. Furthermore, among the secretory signal sequences of glucoamylase, the secretory signal sequence of Rhizopus oryzae-derived GlaR is preferred. Among the secretory signal sequences of β-glucosidase, secretory signal sequences of β-glucosidase (BGL1) and β-glucosidase (BGL7) derived from Aspergillus oryzae are preferable. The endoglucanase secretion signal sequence is preferably an endoglucanase derived from Aspergillus oryzae, a secretory signal sequence of CelA or CelB.

  As the secretory signal sequence, a known secretory signal sequence can be used as it is, or two or more secretory signal sequences can be used in combination. The combination of two or more secretory signal sequences may be a sequence of a plurality of sequences, or may be linked by inserting a short sequence such as a restriction enzyme site between each signal sequence.

  The secretory signal sequence for secreting β-glucosidase outside the yeast cells is preferably a combination of 2 to 5 secretory signal sequences, more preferably a combination of 2 to 3 secretory signal sequences. The secretory signal sequence may be a combination of two or more identical secretory signal sequences, but a combination of different secretory signal sequences is preferred. More preferably, it is a combination of a secretory signal sequence originally possessed by an enzyme secreted outside the yeast cell and other secretory signal sequences. Here, the other secretory signal sequence means any secretory signal sequence having a sequence different from the secretory signal sequence originally possessed by the secreted enzyme.

  The secretory signal sequence for secreting β-glucosidase outside the yeast cell can be used by arbitrarily combining the secretory signal sequences as described above. As a preferable combination, for example, a secretory signal sequence of GlaR and BGL1 secretion signal sequence, GlaR secretion signal sequence and BGL7 secretion signal sequence, GlaR secretion signal sequence and CelA secretion signal sequence, GlaR secretion signal sequence and CelB secretion signal sequence, BGL1 secretion signal sequence and BGL7 secretion signal sequence Secretion signal sequence, secretion signal sequence of BGL1 and secretion signal of CelA, secretion signal sequence of BGL1 and secretion signal sequence of CelB, secretion signal sequence of BGL7 and secretion signal sequence of CelA, secretion signal sequence of BGL7 and secretion signal of CelB Secretion signal sequence, can be mentioned combinations of secretory signal sequences and the like of the secretion signal sequence and CelB of CeIA. More preferable combinations are a combination of a secretion signal sequence of GlaR and a secretion signal of BGL7, and a secretion signal sequence of BGL1 and a secretion signal sequence of BGL7.

  The secretory signal sequence for secreting endoglucanase outside the yeast cell is preferably a combination of the secretory signal sequence inherent in the secreted endoglucanase and the secretory signal sequence of CelA. For example, when secreting the endoglucanase CelB, a combination of the secretory signal sequence of CelB and the secretory signal sequence of CelA is preferable.

When the secretory signal sequence inherent in the secreted enzyme and a heterologous secretory signal sequence are combined, any of the secretory signal sequences may be arranged on the N-terminal side. The secretory signal sequence is preferably N-terminal. The base sequence encoding the secretory signal sequence and the promoter sequence may be continuously connected, and are preferably connected via short sequences such as various restriction enzyme recognition sequences. Examples of restriction enzyme recognition sequences include EcoRI (GAATTC), SalI (GTCGAC), HindIII (AAGCTT), KpnI (GGTACC), PstI (CTGCAG), SacI (GAGCTC), XhoI (CTCGAG), SmaI (CCCGGG), More preferably (GCGGCCGC), the promoter sequence and the secretory signal sequence coding region are linked via a SalI recognition sequence.
Cell surface localized protein The cell surface localized protein may be any protein that is immobilized on, attached to, or adhered to the cell surface and is localized there, and any known protein can be used without limitation.

  Examples of proteins localized in the cell membrane include transmembrane proteins and cell surface localized proteins. The transmembrane protein is a protein that penetrates the lipid bilayer at the hydrophobic amino acid region, and is often found in receptor proteins. On the other hand, a protein modified with a lipid is known as a cell surface localized protein, and this lipid is immobilized on a cell membrane by covalently binding to a membrane component. In addition, there are cell surface localized proteins whose mechanism of immobilization has not been clarified, and these can be used in the method of the present invention. As cell surface localized proteins, various GPI anchoring proteins and BGL2 described later are known. BGL2 is known to bind strongly to the cell wall with yeast β-glucosidase, but its mechanism is unknown. BGL2 does not have a motif common to GPI anchoring proteins.

<GPI anchoring protein>
As a representative example of cell surface localized protein, GPI (glycosylphosphatidylinositol: ethanolamine phosphate-6 mannose α-1,2 mannose α-1,6 mannose α-1,4 glucosamine α-1,6 inositol phospholipid has a basic structure (Glycolipid) anchoring protein. GPI anchoring protein has GPI which is a glycolipid at its C-terminal, and this GPI is bound to the cell membrane surface by covalently binding to PI (phosphatidylinositol) in the cell membrane.

  GPI binding to the C-terminus of the GPI anchoring protein is performed as follows. That is, the GPI anchoring protein is secreted into the endoplasmic reticulum lumen by the action of a secretion signal present on the N-terminal side after transcription and translation. A region called a GPI anchor attachment signal that is recognized when the GPI anchor binds to the GPI anchoring protein exists in the C-terminal region of the GPI anchoring protein or in the vicinity thereof. In the endoplasmic reticulum lumen and the Golgi apparatus, this GPI anchor attachment signal region is cleaved, and GPI binds to the newly generated C-terminus.

  The protein to which GPI is bound is transported to the cell membrane by secretory vesicles, and is fixed to the cell membrane by GPI covalently binding to the PI of the cell membrane. Furthermore, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), and is incorporated into the cell wall so as to be fixed on the cell wall and presented on the cell surface.

  In the present invention, a polynucleotide encoding a normal C-terminal region including a GPI anchor attachment signal region which is a cell membrane binding region of a GPI anchoring protein can be preferably used. This cell membrane-binding region only needs to contain a GPI anchor attachment signal region, and may contain any part of other GPI anchoring protein as long as it does not inhibit the enzyme activity of the fusion protein.

  The GPI anchoring protein may be any protein that functions in yeast cells, and any known GPI anchoring protein can be used without limitation. Known GPI anchoring proteins include, for example, α- or a-agglutinin which is a sex aggregation protein of yeast, Flo1 protein, outer membrane protein OmpA of E. coli (Georgiou, G. et.al. Trends Biotechnol., 11, 6 -10,1993), Escherichia coli maltose transporter LamB, Escherichia coli flagellar protein flagellin, Bacillus subtilis cell wall lytic enzyme CwlB, and the like.

  In particular, yeast α-agglutinin can be preferably used. A region consisting of 320 or 600 amino acids from the C-terminal side of α-agglutinin (ie, a region consisting of 320 amino acids from the C terminus, a region consisting of 321 amino acids from the C terminus, 599 from the C terminus) It is preferable to use a region consisting of amino acids or a region consisting of 600 amino acids from the C-terminal), and more preferably a polynucleotide sequence encoding a region consisting of 320 amino acids from the C-terminal side. In a sequence consisting of 320 amino acids from the C-terminal side of α-agglutinin, there are four sugar chain binding sites. After the GPI anchor is cleaved by PI-PLC, this sugar chain and the polysaccharide constituting the cell wall are covalently bound to enhance the fixation of α-agglutinin to the cell wall. The nucleotide sequence of DNA encoding α-agglutinin is registered with the accession number of CAA89526 in the Japan DNA Data Bank.

In the present invention, the sequence encoding a part of the cell surface localized protein and the GPI anchor attachment signal sequence may be prepared from different sources.
β-glucosidase / endoglucanase β-glucosidase is an enzyme that hydrolyzes a β-glucoside bond between cellooligosaccharide or aglycone and β-D-glucose. The origin of the β-glucosidase of the present invention is not particularly limited, and examples thereof include those derived from Aspergillus oryzae, Aspergillus aculeatus, Saccharomycopsis fibuligera, Bacillus circulans, Clostridium thermocellum, Thermotoga maritima, and Ruminococcus albus. More preferably, β-glucosidase of Aspergillus oryzae is preferable from the viewpoint of safety. The sequence of the β-glucosidase gene of Aspergillus oryzae is registered with the accession number of BAE54829 in the Japan DNA Data Bank.

    Endoglucanase is an enzyme that hydrolyzes the β-glucoside bond of cellulose to produce glucose and cellooligosaccharide. The origin of the endoglucanase of the present invention is not particularly limited, and examples thereof include Aspergillus oryzae, Aspergillus kawachi, Bacillus subtilis, Streptomyces halstedii, Trichoderma reesei, Ruminococcus albus, and Fusarium oxysporum. More preferably, Aspergillus oryzae endoglucanase is preferable from the viewpoint of safety. The sequence of Aspergillus oryzae endoglucanase gene is registered with the accession number of BAA22589 in the Japan DNA Data Bank.

β-glucosidase and endoglucanase may be composed of a part of the amino acid sequence as long as they maintain a practical activity.
Linker In the present invention, the β-glucosidase gene and the downstream cell surface localized protein or a sequence encoding the cell membrane binding region thereof, and the endoglucanase gene and the downstream cell surface localized protein or the cell membrane binding region thereof are combined. A linker is preferably present between the coding sequence. By interposing a linker at the above position, each enzyme activity per cell weight is improved. The “linker” means a polynucleotide encoding a linker peptide.

  In order to improve β-glucosidase activity per cell weight, and thus cellulose degradation rate, and ethanol production rate, between β-glucosidase gene and its downstream cell surface localized protein or sequence encoding its cell membrane binding region The linker length is preferably about 6 to 90 bases, more preferably about 15 to 30 bases. That is, the length of the linker peptide between β-glucosidase and the cell surface localized protein or cell membrane binding region presented on the surface layer of the sake yeast of the present invention is preferably about 2 to 30 amino acid residues, About 10 amino acid residues are more preferable.

  Linker between endoglucanase gene and downstream cell surface localized protein or sequence encoding cell membrane binding region in order to improve endoglucanase activity per cell weight, and thus cellulose degradation rate and ethanol production rate The length is preferably about 6 to 90 bases, more preferably about 42 to 69 bases. That is, the length of the linker peptide between the endoglucanase displayed on the surface layer of the sake yeast of the present invention and the cell surface localized protein or its cell membrane binding region is preferably about 2 to 30 amino acid residues, and 14 to 23 More preferred are amino acid residues.

  The sequence of the linker peptide is not particularly limited, but in order to enhance hydrophilicity, a sequence consisting of amino acids that are uncharged and do not contain an aromatic ring is preferable. Moreover, in order to give flexibility to the linker peptide, an amino acid having a molecular weight of 180 or less and a relatively small bulk is preferable. Examples of such amino acids include glycine, alanine, valine, serine, threonine, and cysteine. Among them, a sequence composed of serine and glycine is preferable for improving the enzyme activity per cell weight.

In the polynucleotides (a) and (b), any oligonucleotide may be used between the secretion signal sequence and the β-glucosidase gene or endoglucanase gene as long as the secretion of the fusion protein by the secretion signal peptide is not inhibited. An array may be inserted. In general, insertion sequences of up to about 30 bases are allowed.
Multi-copy The sake yeast of the present invention preferably contains a plurality of copies of β-glucosidase gene and / or endoglucanase gene in the chromosome. As a result, these enzymes are presented at a high density on the cell surface. However, in order not to degrade β- (1,4) glucan or β- (1,6) glucan in the cell wall of yeast itself, the upper limit of the copy number of each gene is usually about 6.
Method for producing sake yeast of the present invention The sake yeast of the present invention is inserted into a chromosome integration type expression vector so that the polynucleotides (a) and (b) are expressed by respective promoters, and the plasmid is And can be obtained by transforming sake yeast. As long as the expression vector does not contain an autonomously replicating sequence of yeast, it is usually a yeast chromosomal DNA integration type.

  The promoter is not particularly limited as long as it can function in yeast cells. For example, yeast SED1 promoter (Japanese Patent Laid-Open No. 2003-265177), GAPDH promoter (JP 07-24594), PGK1 promoter (EMBO J. (1982), 1,603- Tuite, MF et al), ADH1 promoter (Nature (1981) , 293, 717- Hitzeman, RA et al). These promoters exhibit high promoter activity in yeast cells.

  When culturing on an industrial scale, a reaction system in which various components are dissolved, that is, a high osmotic pressure environment is often obtained. In addition, it is difficult to strictly control the temperature and pH compared to the case of culturing in a laboratory, and it is difficult to use a nutrient rich culture medium in terms of cost and the like. In this regard, the yeast SED1 promoter not only shows high transcriptional activity under normal culture conditions, but also stable and high in various stress environments such as high temperature, high osmotic pressure or low osmotic pressure, oligotrophic state, presence of alcohol, etc. This is particularly preferable because it exhibits promoter activity and is not easily affected by pH fluctuations.

  The promoter may be the entire region or a partial region as long as it exhibits high promoter activity.

  Further, in order to regulate the expression of β-glucosidase gene and endoglucanase gene, an operator, an enhancer, a terminator and the like may be included in addition to the promoter. Examples of the terminator include ADH1 (aldehyde dehydrogenase) terminator, GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) terminator, and the like.

  Examples of an expression vector having a GAPDH promoter that can be incorporated into yeast chromosomal DNA include pICAS1 (sold by Professor Mitsumi Ueda, Graduate School of Agriculture, Kyoto University). An expression vector containing SED1 promoter, PGK1 promoter, or ADH1 promoter can be prepared by amplifying these promoters by PCR and ligating them to plasmid pRS406 (Stratagene).

  An arbitrary nucleotide sequence may be inserted between the promoter and the secretory signal sequence as long as the fusion protein expression by the promoter is not inhibited.

  Sake yeast in which a plurality of copies of β-glucosidase gene and endoglucanase gene are incorporated in the chromosome is, for example, an expression vector containing both (a) and (b), and different markers (for example, drug resistance genes) , URA3, TRP1, LEU2, LYS2, etc.) can be used by transforming sake yeast using a plurality of those. In addition, transforming sake yeast using an expression vector containing both (a) and (b) and containing a drug resistance gene as a selectable marker, the drug concentration changed stepwise or gradually The transformant in which a plurality of the expression vectors are introduced can also be easily selected. In addition, the number of genes to be introduced into the host can be determined by setting the number of genes to be inserted into the expression vector.

  The number of copies of each gene introduced into the host can be determined by measuring the enzyme activity secreted by the transformant, genomic Southern hybridization using probes specific to each gene, PCR using primers specific to each gene, etc. Can be confirmed.

It can be confirmed by a conventional method that β-glucosidase and endoglucanase are immobilized on the cell surface of the obtained yeast. For example, a method in which an antibody against these proteins and a fluorescently labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase are allowed to act on a test yeast, an antibody against these proteins and a biotin-labeled secondary antibody Examples include a method of reacting an antibody with a fluorescent labeled streptavidin after reacting with the antibody. In addition, when a FLAG tag is inserted on the N-terminal side of the anchor protein, it can be confirmed that localization on the cell surface layer is simpler by using a FLAG tag antibody.
<Host>
In the present invention, sake yeast having high fermentability, high ethanol tolerance and genetically stable is used among practical yeasts. In general, ethanol is toxic to microorganisms. Yeast that undergoes ethanol fermentation is no exception, and when the ethanol concentration exceeds 8% (v / v), it is gradually killed by the ethanol it produces. Here, sake yeast is a strain that has been selected and bred for many years with sake moromi with an ethanol concentration as high as 20%, and has extremely high ethanol resistance compared to general yeast.

Examples of sake yeast include “Kyokai Yeast” distributed by Japan Brewing Association and mutant strains using these as parent strains. In addition, any yeast that is used in sake brewing is useful. When an auxotrophic gene is used as a transformation marker, a strain obtained by imparting auxotrophy to these sake yeasts using a technique such as mutation may be used. Such auxotrophs include uracil auxotrophy and tryptophan. Requirement, leucine requirement, histidine requirement, lysine requirement and the like. Saccharomyces cerevisiae GRI-117-UK obtained by mutagenesis using “Kyokai No. 9 yeast” as an example of conferring uracil / lysine requirement to sake yeast.
(II) Method for Producing Ethanol The method for producing ethanol of the present invention comprises degrading a polymer formed by β-glucoside bonds of glucose in the presence of the sake yeast of the present invention to obtain glucose, and decomposing the produced glucose. This is a method for obtaining ethanol.
Polymer by β-glucoside bond of glucose The degree of polymerization of the polymer by β-glucoside bond of glucose and the mode of glucoside bond are not particularly limited. For example, oligosaccharide such as cellooligosaccharide, cellulose, and β-glucan Is mentioned.

These polymers can be obtained from known raw materials such as acid treatment and supercritical treatment from raw biomass such as waste biomass, waste biomass such as food waste containing dietary fiber, or resource crop biomass. Or by treating them under milder conditions in these methods. According to this method, usually, a mixture of β-glucan and its partially decomposed product is obtained. This may be used as it is, β-glucan and a partial decomposition product thereof may be used after purification, or only a specific β-glucose polymer may be used after purification.
Decomposition reaction The decomposition reaction of the polymer can be carried out in a state where yeast is suspended in the reaction solution in the reaction vessel. Moreover, it can also carry out using the bioreactor which filled yeast in reaction containers like a column. The reaction may be carried out by any of continuous, batch (batch) and semi-batch methods.

  The reaction solution may contain only a β-glucose polymer as a substrate, or in addition to this, a buffer for pH adjustment or a known substance necessary for the survival of yeast may be contained. Good. Preferably, a mixture obtained by acid treatment or supercritical treatment of plant material may be used as the substrate solution.

  The β-glucose polymer concentration at the start of the reaction is usually preferably about 1 to 20% by weight, more preferably about 5 to 15% by weight. Within the above concentration range, glucose can be produced efficiently, and the viscosity of the reaction solution increases, making stirring and temperature control difficult, or the osmotic pressure of the reaction solution increases to inhibit yeast death and growth. Will not occur.

  The reaction temperature should just be the temperature which each enzyme which sake yeast hold | maintains activity, and should just be about 30-38 degreeC normally.

By monitoring the concentration of each component in the reaction solution over time using, for example, gas chromatography or HPLC, the additional amount of cellulose, the reaction time, the added amount of the pH adjuster, etc. may be determined. If it is said temperature range, ethanol will produce | generate in a reaction liquid by reaction for about 5 to 24 hours.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Construction of expression vector containing β-glucosidase and endoglucanase
(A) A highly active promoter (SED1 promoter) was obtained according to a conventional method. Briefly, yeast Saccharomyces cerevisiae X2180 using two primers 5′-GCGggatccTTGGATATAGAAAATTAACGTAAGG-3 ′ (SEQ ID NO: 1 Psed-F) and 5′-CCGgaattcCTTAATAGAGCGAACGTATTTTATT-3 ′ (SEQ ID NO 2: Psed-R). PCR was performed using -1A (ATCC Number: 204647) chromosomal DNA as a template. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times.

  Here, SEQ ID NO: 1: Psed-F is given a recognition sequence for restriction enzyme KpnI for the purpose of later insertion into pRS406, and an extra sequence for increasing the efficiency of restriction enzyme treatment. For SEQ ID NO: 2: Psed-R, a restriction enzyme EcoRI recognition sequence and an extra sequence are added for the same purpose. Restriction enzyme recognition sequences are shown in lower case.

  The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then digested with restriction enzymes KpnI and EcoRI to obtain KpnI and EcoRI fragments. This KpnI and EcoRI fragment contains the promoter region of the SED1 gene. The sequence is shown in SEQ ID NO: 3 (Psed-seq) in the Sequence Listing.

(B) A secretory signal sequence of Rhizopus oryzae glucoamylase was obtained according to a conventional method. Briefly, 5′-GCGggatcc ATGCAACTGTTCAATTTGCCATTGA -3 ′ (SEQ ID NO: 4: sig-F) 5′-ggggttaacgtcgacgatctccgcgGCAGAAACGAGCAAAGAAAAGTAAG-3 ′ (SEQ ID NO: 5: sig-R) was synthesized as a primer and the glucoamylase gene PCR was performed using the plasmid pNGB1 (Gene 207 127-134 (1998)) into which is inserted as a template. As PCR conditions, a cycle of 94 ° C./1 minute-52 ° C./1 minute-72 ° C./30 seconds was repeated 30 times.

  Here, SEQ ID NO: 4: sig-F is given a restriction enzyme EcoRI recognition sequence for later insertion into pRS406, and an extra sequence for increasing the efficiency of restriction enzyme treatment. SEQ ID NO: 5: The purpose of designing sig-R is to provide recognition sites for restriction enzymes SalI and HpaI in order to insert the target gene sequence later, and to simplify restriction enzyme treatment prior to insertion into pRS406. Then, the sequence after restriction enzyme SmaI digestion was added to the end of the sequence. Restriction enzyme recognition sequences are shown in lower case.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with the restriction enzyme EcoRI to obtain an EcoRI-SmaI fragment. This EcoRI fragment contains a glucoamylase secretion signal sequence. The sequence is shown in (SEQ ID NO: 6: sig-seq) in the sequence listing.
(C) Chromosomal DNA is obtained from yeast Saccharomyces cerevisiae X2180-1A by a conventional method in order to obtain a sequence having a gene (320 amino acids) having a sequence encoding a C-terminal part of α-agglutinin and a GPI anchor attachment signal sequence. And 5′-gggGctcgagCGCCAAAAGCTCTTTTATCTCAA-3 ′ (SEQ ID NO: 7: GPI-F) and 5′-AAGGAAAAAAgcggccgcTTTGATTATGTTCTTTCTATTTGAATG-3 ′ (SEQ ID NO: 8: GPI-R) were used for PCR. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times.

  Here, for the design of SEQ ID NO: 7: GPI-F, in order to insert a target linker sequence later, a recognition site for a restriction enzyme XhoI is added, and the restriction enzyme treatment before insertion into pRS406 is simplified. Then, the sequence after restriction enzyme SmaI digestion was added to the end of the sequence. SEQ ID NO: 8: GPI-R is given a restriction enzyme NotI recognition sequence for later insertion into pRS406, and an extra sequence for efficient restriction enzyme treatment. Restriction enzyme recognition sequences are shown in lower case.

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and digested with restriction enzyme NotI to obtain a SmaI-NotI fragment. This SmaI-NotI fragment contains a sequence encoding 320 amino acids from the C-terminus of α-agglutinin and 466 bp of the 3 ′ non-coding region, and a GPI anchor attachment signal sequence is included in this sequence. . The sequence is shown in SEQ ID NO: 9: GPI-seq in the Sequence Listing.

(D) As a basic vector used for yeast surface display expression, a highly active promoter obtained in (A), a secretory signal sequence obtained in (B), and (C) a DNA fragment containing a GPI anchor attachment signal, The plasmid pK113 was sequentially inserted into the multicloning site, KpnI-EcoRI site, EcoRI-SmaI site and SmaI-NotI site in pRS406, respectively.

(E) Aspergillus oryzae β-glucosidase gene cDNA was obtained according to a conventional method. In brief, first, Aspergillus oryzae O-1013 strain (National Institute of Advanced Industrial Science and Technology patent biological deposit center on November 20, 1997 (Tsukuba Center Central 6th 1-1-1, Tsukuba, Ibaraki, Japan) Total RNA was extracted from FERM P-16528, and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. That is, 5′-ACGCgtcgacATGAAGCTTGGTTGGATCGAGGTGG-3 ′ (SEQ ID NO: 10: B-F) and 5′-aacCCTGGGCCTTAGGCAGCGACGCCTGGAGCGGCAG-3 ′ (SEQ ID NO: 11: B-R) were synthesized, and PCR was carried out using this cDNA mixture as a template. PCR was performed under the conditions of repeating a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 2 minutes 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes.

  Here, in SEQ ID NO: 10: BF, a restriction enzyme SalI recognition sequence is used immediately before the translation initiation codon ATG to fuse the N-terminus of the gene with a secretory signal sequence, and the restriction enzyme treatment is made efficient. An extra array of is assigned. In SEQ ID NO: 11 BR, the translation termination codon is removed to later fuse the C terminus of the gene with the N terminus of the α-agglutinin C terminus fragment, and restriction enzyme treatment prior to insertion into pK113 is simplified. For this purpose, a sequence after restriction enzyme HpaI digestion was added to the end of the sequence. Restriction enzyme recognition sequences are shown in lower case.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit), and then cleaved with the restriction enzyme SalI to obtain an approximately 2.6 kbp β-glucosidase gene cDNA fragment. The sequence is shown in (SEQ ID NO: 12: B-cDNA) in the sequence listing.
(F) The obtained target DNA was inserted into the SalI and SmaI cleavage sites of the plasmid pK113 to obtain the target plasmid pK113-BGL7.

(G) For the endoglucanase gene, 5′-GTAAgtcgacATGATCTGGACACTCGCTC-3 ′ (SEQ ID NO: 13: CF) and 5′-CCCAgttaacCATGCCTGTAGGTAGATCCA-3 ′ (SEQ ID NO: 14: CR) are used according to the same procedure as in (E). An about 1.3 kbp endoglucanase gene cDNA fragment was prepared. Subsequently, the SalI restriction enzyme site existing in the endoglucanase gene sequence was 5′-TTCGATGTtGAtGCCTCCACCCTC-3 ′ (SEQ ID NO: 15: C-dS1F), 5′-GTGGAGGCaTCaACATCGAAGGTGAAT-3 ′ (SEQ ID NO: 16: C- dS1R), 5'-GAAAAACTGTtGAtTCAATCACAAAGGACT-3 '(SEQ ID NO: 17: C-dS2F) and 5'-TTGTGATTGAaTCaACAGTTTTTCCTCCAG-3' (SEQ ID NO: 18: C-dS2R) Destroyed. The sequence is shown in (SEQ ID NO: 19: C-cDNA) in the sequence listing. In SEQ ID NO: 19, the part subjected to base substitution is shown in small letters.
(H) The obtained target DNA was inserted into the SalI and SmaI cleavage sites of plasmid pK113 to obtain the desired plasmid pK113-celB.

Examination of the effect of linker length on enzyme activity
(1) Plasmid construction In the plasmid (pK113-BGL7) obtained in Example 1, the recognition site of the restriction enzyme XhoI existing between the β-glucosidase gene and the sequence encoding the C-terminal region of α-agglutinin is restricted. 18 bases (6 amino acids; linker L6), 27 bases (9 amino acids; linker L9), 42 bases (14 amino acids; linker L14), 54 bases (18 amino acids; linker L18) synthesized by cleaving with the enzyme XhoI, By inserting a linker of 69 bases (23 amino acids; linker L23), a plasmid into which the linker of each length was inserted, pK113-BGL7 (L6), pK113-BGL7 (L9), pK113-BGL7 (L14), pK113-BGL7 (L18) and pK113-BGL7 (L23) were prepared.

  Further, in the plasmid (pK113-celB) obtained in Example 1, plasmids pK113-celB (L6), pK113-celB (L9), pK113-celB (L14), pK113-celB (L18), And pK113-celB (L23) was prepared.

The base sequence of each linker and the amino acid sequence of the corresponding linker peptide are as follows. At the time of insertion into pK113-BGL7 and pK113-celB, a base sequence corresponding to the sense strand and the antisense strand is prepared by adding a sequence after treatment with XhoI restriction enzyme at both ends, and annealing them to each plasmid. An insert into was made.
<Linker L6>
Base sequence: GGTTCTTCAGGTGGTTCG (SEQ ID NO: 20)
Amino acid sequence: GSSGGS (SEQ ID NO: 21)
<Linker L9>
Base sequence: GGTTCGAGTGGTTCTTCAGGTGGTTCG (SEQ ID NO: 22)
Amino acid sequence: GSSGSSGGS (SEQ ID NO: 23)
<Linker L14>
Base sequence: GGTTCGAGTGGTTCTTCAGGTGGTTCTGGTGGATCTGGTTCG (SEQ ID NO: 24)
Amino acid sequence: GSSGSSGGSGGSGS (SEQ ID NO: 25)
<Linker L18>
Base sequence:
GGTTCTTCAGGTGGTTCGAGTGGTTCTTCAGGTGGTTCTGGTGGATCTGGTTCG (SEQ ID NO: 26)
Amino acid sequence: GSSGGSSGSSGGSGGSGS (SEQ ID NO: 27)
<Linker L23>
Base sequence:
GGTTCTTCAGGTGGTTCTGGTGGATCTGGTTCGAGTGGTTCTTCAGGTGGTTCTGGTGGATCTGGTTCG (SEQ ID NO: 28)
Amino acid sequence: GSSGGSGGSGSSGSSGGSGGSGS (SEQ ID NO: 29)

(2) Transformation Using these plasmids, sake yeast No. 9 is described in a known method, more specifically, in the “Yeast Genetic Experiment Manual (Maruzen Co., Ltd.) P. 9 to P. 17”. The GRI-117-UK strain having both uracil requirement and lysine requirement was obtained (owned by Laurel Wreath Co., Ltd.). Using this strain as a host, pK113-BGL7 and pK113-celB were transformed by the lithium acetate method, respectively, and sake yeast with 2 copies of β-glucosidase gene inserted in the chromosome and 2 copies of endo in the chromosome Sake yeast into which glucanase was inserted was prepared.

(3) Measurement of β-glucosidase activity
Each yeast was cultured at 30 ° C. for 24 hours using 5 ml of SD medium (ΔURA). The preculture was inoculated into 5 ml of YPD medium so that the OD ₆₀₀ was 0.1 per 5 ml, and cultured with shaking at 30 ° C. for 48 hours. The cells were collected by centrifuging at 5000 × g for 5 minutes, washed with sterilized water, and then collected by the same centrifugation. 0.4 ml of a substrate solution (1 mM p-nitrophenyl β-D-glucopyranoside (sigma) aqueous solution (pH 5.0)) was added to the cells. The OD _{600 at} this time is about 0.5. After further reaction at 37 ° C. for 10 minutes, 0.4 ml of 0.5 M Na ₂ CO ₃ was added, and the amount of p-nitrophenol released was quantified by measuring the absorbance at 415 nm. The activity produced by 1 μmol of p-nitrophenol per minute was defined as 1U.

(4) Measurement of endoglucanase activity
Each yeast was cultured at 30 ° C. for 24 hours using 5 ml of SD medium (ΔURA). The preculture was inoculated into 5 ml of YPD medium so that the OD ₆₀₀ was 0.1 per 5 ml, and cultured with shaking at 30 ° C. for 48 hours. The cells were collected by centrifuging at 5000 × g for 5 minutes, washed with sterilized water, and then collected by the same centrifugation. 0.5 ml of a substrate solution (0.1% by weight barley β-glucan (sigma) aqueous solution (pH 4.0)) was added to the cells. The OD _{600 at} this time is about 10. After further reaction at 40 ° C. for 30 minutes, the mixture was centrifuged at 15000 × g for 10 minutes, and the reducing sugar concentration in the supernatant was measured according to the Somogi-Nelson method. The activity for producing 1 μmol of reducing sugar per minute was defined as 1U.

(5) Results The results are shown in FIG. From FIG. 1, the β-glucosidase activity per dry weight of the bacterial cells is obtained when a linker peptide consisting of 6 to 9 amino acid residues is interposed between β-glucosidase and α-agglutinin, and when the linker peptide is not included. It can be seen that the increase is about 6 to 7 times.

  Further, the endoglucanase activity per dry weight of the microbial cells is about 5 when the linker peptide consisting of 18 amino acid residues is interposed between the endoglucanase and α-agglutinin, compared to the case where the linker peptide is not included. It can be seen that it doubles.

  In "Science and Industry" Vol. 78 (6), p16-21 (2004), when presenting the lipase of the filamentous fungus Rhizopus oryzae on the cell surface of the yeast Saccharomyces cerevisiae, the C of lipase and α-agglutinin It is described that the lipase activity was doubled by interposing a 7 to 14 amino acid linker peptide consisting of serine and glycine between the terminal region (page 19, Table 1). In the present invention, the effect of increasing the enzyme activity by inserting the linker is much higher than this.

Examination of the effect of secretory signal sequence on enzyme activity
(1) Construction of plasmids Several plasmids were prepared by replacing the secretion signal sequence of the β-glucosidase gene in the plasmid (pK113-BGL7) obtained in Example 1. Specifically, a product in which one or two secretory signal sequences of Aspergillus oryzae-derived β-glucosidase (BGL1, BGL7) and Rhizopus oryzae-derived glucoamylase (GlaR) were linked was prepared. At this time, the restriction enzyme site immediately before the translation initiation codon was changed from EcoRI to SalI. Each secretory signal sequence was obtained by PCR using A. oryzae Poly (A) + RNA or plasmid pNGB1 as a template according to the same procedure as in Example 1, and ligated to the BGL7 prosequence. Then, plasmids pK113-BGL7 (d7), pK113-BGL7 (d1V7), pK113-BGL7 (d7D7), and pK113-BGL7 (dR7) in which the secretory signal sequence was substituted were prepared. The plasmid (pK113-BGL7) obtained in Example 1 has a secretion signal sequence of GlaR and a secretion signal sequence of BGL7. The BGL7 mature sequence is shown below.

<Base sequence>
(SEQ ID NO: 34)

<Amino acid sequence>
(SEQ ID NO: 35)

  Similarly, several plasmids in which the secretion signal sequence of the endoglucanase gene was changed were prepared for the plasmid of Example 1 (pK113-celB). Specifically, a product in which one or two secretory signal sequences of Aspergillus oryzae-derived endoglucanase (CelA, CelB) and Rhizopus oryzae-derived glucoamylase (GlaR) were linked was prepared. At this time, the restriction enzyme site immediately before the translation initiation codon was changed from EcoRI to SalI. Each secretory signal sequence was obtained by PCR using A. oryzae Poly (A) + RNA or plasmid pNGB1 as a template according to the same procedure as in Example 1, and ligated to the celB mature sequence. Then, plasmids pK113-celB (dB), pK113-celB (dA2B), pK113-celB (dB2B), and pK113-celB (dRB) in which the secretory signal sequence was substituted were prepared. The celB mature sequence is shown below.

<Base sequence>
(SEQ ID NO: 36)

<Amino acid sequence>
QVGTTADAHPRLTTYKCTSQNGCTRQNTSVVLDAATHFIHKKGTQTSCTNSNGLDTAICPDKQTCADNCVVDGITDYASYGVQTKNDTLTLQQYLQTGNATKSLSPRVYLLAEDGENYSMLKLLNQEFTFDVDASTLVCGMNGALYLSEMEASGGKSSLNQAGAKYGTGYCDAQCYTTPWINGEGNTESVGSCCQEMDIWEANARATGLTPHPCNTTGLYECSGSGCGDSGVCDKAGCGFNPYGLGAKDYYGYGLKVNTNETFTVVTQFLTNDNTTSGQLSEIRRLYIQNGQVIQNAAVTSGGKTVDSITKDFCSGEGSAFNRLGGLEEMGHALGRGMVLALSIWNDAGSFMQWLDGGSAGPCNATEGNPALIEKLYPDTHVKFSKIRWGDIGSTYRHG (SEQ ID NO: 37)

The name of each secretion signal: component, base sequence, and amino acid sequence corresponding thereto are as follows.
<β-Glucosidase>
d7: Secretory signal sequence of BGL7, ATGAAGCTTGGTTGGATCGAGGTGGCCGCATTGGCGGCTGCCTCAGTAGTCAGTGCCAAG (SEQ ID NO: 38), MKLGWIEVAALAAASVVSAK (SEQ ID NO: 39)
d1V7: Concatenate BGL1 secretion signal sequence and BGL7 secretion signal sequence, ATGAAGCTGTCAGCGGCACTTTCTACGCTTGCTGCATTGCAGCCAGCAGTCGGTGCTGCTGTTATGAAACTTGGTTGGATCGAGGTGGCCGCATTGGCGGCTGCCTCAGTAGTCAGTGCCQP
dR7: Concatenate GlaR secretion signal sequence and BGL7 secretion signal sequence, ATGCAACTGTTCAATTTGCCATTGAAAGTTTCATTCTTTCTCGTCCTCTCTTACTTTTCTTTGCTCGTTTCTGCTGCTGAAATTATGAAACTTGAKTGGATCGAGGTGGCCGCATTGGCGGCTGCCTCAGTAGTCAGTGLK VS LV

<Endoglucanase>
dB: Secret signal sequence of CelB, ATGATCTGGACACTCGCTCCCTTTGTGGCACTCCTGCCACTGGTAACTGCCCAG (SEQ ID NO: 44), MIWTLAPFVALLPLVTAQ (SEQ ID NO: 45)
dB2B: Concatenate two secret signal sequences of CelB, ATGATCTGGACACTCGCTCCCTTTGTGGCACTCCTGCCACTGGTAACTGCTCAACAAATGATTTGGACACTCGCTCCCTTTGTGGCACTCCTGCCACTGGTAACTGCCCAG (SEQ ID NO: 46), MIWTLAPFVALLPLVTAQQMIWTLAPFVALLPLVTAQ (SEQ ID NO: 47)
dA2B: Concatenate CelA secretion signal sequence and CelB secretion signal sequence, ATGAAGCTCTCATTGGCACTTGCTACGCTCGTGGCCACAGCATTCAGTCAAGAGATGATCTGGACACTCGCTCCCTTTGTGGCACTCCTGCCACTGGTAACTGCCCAG (SEQ ID NO: 48), MKLSLALATLVATAFSQEMIWTLAPFVALLPQQ
dRB: Concatenate GlaR secretion signal sequence and CelB secretion signal sequence, ATGCAACTGTTCAATTTGCCATTGAAAGTTTCATTCTTTCTCGTCCTCTCTTACTTTTCTTTGCTCGTTTCTGCTGCTGAAATTATGATCTGGACACTCGCTCCCTTTGTGGCACTCCTGCCACTGGTAACTGCCCAG (SEQ ID NO: FSLKLSLV

(2) Transformation
In the same manner as in Example 2, sake yeast in which two copies of β-glucosidase gene were incorporated in the chromosome and sake yeast in which two copies of endoglucanase were incorporated in the chromosome were prepared.

(3) Measurement of β-glucosidase activity
Each yeast was cultured at 30 ° C. for 24 hours using 5 ml of SD medium (ΔURA). The preculture was inoculated into 5 ml of SDC medium (ΔURA, 2% by weight casamino acid) so that the OD ₆₀₀ was 0.1 per 5 ml, and cultured with shaking at 30 ° C. for 64 hours. The cells were collected by centrifuging at 5000 × g for 5 minutes, washed with sterilized water, and then collected by the same centrifugation. 0.4 ml of a substrate solution (1 mM p-nitrophenyl β-D-glucopyranoside (sigma) aqueous solution (pH 5.0)) was added to the cells. The OD _{600 at} this time is 0.5. After further reaction at 37 ° C. for 3 minutes, 0.4 ml of 0.5 M Na ₂ CO ₃ was added, and the amount of p-nitrophenol released was quantified by measuring the absorbance at 415 nm. The activity produced by 1 μmol of p-nitrophenol per minute was defined as 1U.

(4) Measurement of endoglucanase activity
Each yeast was cultured at 30 ° C. for 24 hours using 5 ml of SD medium (ΔURA). The preculture was inoculated into 5 ml of SDC medium (ΔURA, 2% by weight casamino acid) so that the OD ₆₀₀ was 0.1 per 5 ml, and cultured with shaking at 30 ° C. for 64 hours. The cells were collected by centrifuging at 5000 × g for 5 minutes, washed with sterilized water, and then collected by the same centrifugation. After adding 0.2 ml of 50 mM acetic acid-sodium acetate buffer solution (pH 4.5) to this microbial cell suspension, the substrate solution (2 wt% AZO-CM-Cellulose (Megazyme) aqueous solution (pH 4.5)) 0.2 ml was added. The OD _{600 at} this time is 25. After further reaction at 37 ° C for 15 minutes, 1 ml of a reaction stop solution (2.4% sodium acetate, 0.4% zinc acetate, 80% ethanol (pH 5.0)) was added and stirred, and then centrifuged at 15000 xg for 10 minutes. The absorbance at 590 nm of the supernatant was measured. The activity to increase the absorbance at 590 nm by 1 per minute was defined as 1U.

(5) Results The results are shown in FIG. As shown in FIG. 2, the β-glucosidase activity per dry weight of the bacterial cells is changed from the restriction enzyme EcoRI recognition sequence to the SalI recognition sequence by changing the sequence immediately before the start codon so that the enzyme activity on the yeast cell surface is the strain of Example 1. It was found to improve about 1.7 times compared to. It was clarified that a combination of the secretion signal of GlaR and the secretion signal of BGL7 is the optimal secretion signal sequence that maximizes the activity of BGL7.

  The endoglucanase activity per dry weight of the cells is highest when the sequence immediately before the start codon is changed from the restriction enzyme EcoRI recognition sequence to the SalI recognition sequence and the secretion signal of CelA and the secretion signal of CelB are linked. As compared with the strain of Example 1, the enzyme activity in the yeast cell surface layer was improved by about 6.5 times.

Examination of the effect of optimization of linker length and secretory signal sequence on enzyme activity
(1) Construction of plasmid Based on the results obtained in Examples 2 and 3, a plasmid expressing β-glucosidase in which both the linker length and the secretory signal sequence were optimized was prepared. Specifically, the sequence immediately before the start codon is a SalI recognition sequence that links the secretion signal of GlaR and the secretion signal of BGL7, and a linker peptide consisting of 6 amino acid residues is interposed between β-glucosidase and α-agglutinin. Plasmid pK113-BGL7 (dR7-L6) was prepared according to a conventional method.

Similarly, a plasmid that expresses endoglucanase with optimized linker length and secretory signal sequence was prepared. Specifically, the sequence immediately before the start codon is a SalI recognition sequence that links the secretion signal of CelA and the secretion signal of CelB, and a linker peptide consisting of 18 amino acid residues is interposed between endoglucanase and α-agglutinin. Plasmid pK113-celB (dA2B-L18) was prepared according to a conventional method.

(2) Transformation
In the same manner as in Example 2, sake yeast in which two copies of β-glucosidase gene were incorporated in the chromosome and sake yeast in which two copies of endoglucanase were incorporated in the chromosome were prepared.
(3) Measurement of β-glucosidase activity
The same operation as in Example 3 was performed.
(4) Measurement of endoglucanase activity
The same operation as in Example 3 was performed.
(5) Results The results are shown in FIG. From FIG. 3, β-glucosidase activity and endoglucanase activity per dry weight of the bacterial cells were improved by 3.2 times and 8.6 times, respectively, before optimization by optimizing both the linker length and the secretory signal sequence.

Ethanol fermentation from β-glucan A medium containing 6.7 g / l YNB w / o amino acids, 20 g / l casamino acids, 20 g / l β-glucan as a single carbon source in Examples 1 and 4 Add each BGL7-expressing strain / CelB-expressing strain to OD ₆₆₀ = 5 (total microbial mass: OD ₆₆₀ = 10) and measure the ethanol concentration when reacted at 30 ° C by gas chromatography did. At this time, as a comparative control, a parent strain that does not display the enzyme on the cell surface and a medium that uses glucose as a single carbon source instead of β-glucan (6.7 g / l YNB w / o amino acids, 20 g / l casamino acids, 20 g / l glucose) was prepared.

The result is shown in FIG. The parent strain could not produce ethanol from β-glucan medium, whereas the BGL7 expression strain / CelB expression strain mixed system did ethanol fermentation well. The fermentation rate at this time was the largest in a system in which a strain in which both the linker length and the secretion signal sequence were optimized was about 70% of the ethanol production rate from glucose. From this, it can be expected that if the production strain of the present invention is used, ethanol can be produced at a rate equivalent to glucose from a cellulose degradation product obtained from a plant material by a known hydrolysis treatment method.

In addition, it was confirmed that about 0.9% of ethanol was produced in the strain before optimization after 24 hours of reaction, and that this strain can be used sufficiently for ethanol production.

Ethanol fermentation from β-glucan
(1) Construction of expression vector containing β-glucosidase and endoglucanase
(A) A plasmid pUDB7CB containing a uracil marker, β-glucosidase, and endoglucanase was prepared. Specifically, 2 primers of 5′-CCGCgacgtcTTGGATATAGAAAATTAACG-3 ′ (SEQ ID NO: 30: P-SED1 (AatII) F) and 5′-CCGCgacgtcTTTGATTATGTTCTTTCTAT-3 ′ (SEQ ID NO: 31: CAS1 (AatII) R) are used. Thus, PCR was performed on the portion corresponding to the terminator from the promoter using pK113-celB prepared in Example 1, and a PCR product encoding an endoglucanase-expressing synthetic gene was obtained. The obtained PCR product was digested with the restriction enzyme AatII and purified. PK113-BGL7 prepared in Example 1 was digested with the restriction enzyme AatII, and the above endoglucanase expression synthetic gene was inserted into the cleavage site.

(B) Plasmid pUDB7CBII containing uracil marker, linker length optimized β-glucosidase, and linker length optimized endoglucanase was constructed. Specifically, pK113-celB (L18) prepared in Example 2 was used instead of pK113-celB, and pK113-BGL7 (L6) prepared in Example 2 was used instead of pK113-BGL7. (A) It was produced by the same method.

(C) Plasmid pKDB7 containing a lysine marker and β-glucosidase was prepared. Specifically, using two primers of 5′-GCGgacgtcCACTTGCAATTACATA-3 ′ (SEQ ID NO: 32: LYS2 (AatII) F) and 5′-CTGCACGTGATTTACAGTTCTTATTCAATA-3 ′ (SEQ ID NO: 33: LYS2 (Blunt) R), PCR was performed using the chromosomal DNA of the yeast Saccharomyces cerevisiae X2180-1A as a template to obtain a PCR product encoding the LYS2 gene. The obtained PCR product was digested with the restriction enzyme AatII and purified. PK113-BGL7 prepared in Example 1 was digested with restriction enzyme AatII and restriction enzyme NaeI, and the LYS2 gene was inserted into the cleavage site.

(D) A plasmid pKDB7 (L6) containing a lysine marker and linker-length optimized β-glucosidase was prepared. Specifically, pK113-BGL7 (L6) prepared in Example 2 was used instead of pK113-BGL7, and the same method as in (C) was used.

(E) Plasmid pKDB7CBII containing lysine marker, linker length optimized β-glucosidase, and linker length optimized endoglucanase was constructed. Specifically, pK113-celB (L18) prepared in Example 2 was used instead of pK113-celB, and pKDB7 (L6) prepared in (D) was used instead of pK113-BGL7. It was produced by the method of.

(2) Preparation of surface display yeast Using the lithium acetate method, pUDB7CB and pKDB7 were transformed into host GRI-117-UK, and 4 copies of β-glucosidase gene and 2 copies of endoglucanase gene were incorporated into the chromosome. Yeast UK / UDB7CB / KDB7 strains containing sake yeast and no linker were prepared.

    Similarly, using pUDB7CBII and pKDB7 (L6), 4 copies of the β-glucosidase gene and 2 copies of the endoglucanase gene are contained in the chromosome, and 6 between the β-glucosidase gene and the C-terminal region of α-agglutinin. A yeast UK / UDB7CBII / KDB7 (L6) strain containing an amino acid residue linker peptide and an 18 amino acid residue linker peptide between the endoglucanase gene and the C-terminal region of α-agglutinin was prepared.

    Similarly, sake yeast containing 4 copies of β-glucosidase gene and 4 copies of endoglucanase gene in the chromosome using pUDB7CBII and pKDB7CBII, and between the β-glucanase gene and the C-terminal region of α-agglutinin A yeast UK / UDB7CBII / KDB7CBII strain containing a linker peptide of 6 amino acid residues and a linker peptide of 18 amino acid residues between the endoglucanase gene and the C-terminal region of α-agglutinin was prepared.

Similarly, sake yeast UK / UDB7 strain in which two copies of β-glucosidase gene were inserted into the chromosome was prepared using pK113-BGL7. In addition, using pK113-celB, a sake yeast UK / UDCB strain in which two copies of the endoglucanase gene were inserted into the chromosome was prepared.
(3) Ethanol fermentation using β-glucan as a raw material Each of the transformants obtained as described above and the sake yeast GRI-117-UK as a host were inoculated into 5 ml of YPD medium at 30 ° C. Pre-cultured for 16 hours. This was added to 100 ml of YPD medium and cultured with shaking at 30 ° C. for 30 hours. The cells were collected by centrifugation at 5,000 × g for 5 minutes, and suspended in a 2% barley β-glucan (sigma) aqueous solution (pH 5.0) so that the OD ₆₀₀ was 5.

  This was reacted at 37 ° C. for 16 hours, and then the ethanol concentration in the bacterial solution was measured by gas chromatography.

  The results are shown in FIG. From FIG. 5, the ethanol fermentation using the co-presenting yeast is more effective per dry weight of the cells than the case where the ethanol fermentation is performed using the yeast displaying the β-glucosidase on the surface and the yeast displaying the endoglucanase on the surface. It can be seen that the ethanol production rate is high. Furthermore, it can be seen that the ethanol production rate is further improved by interposing a linker peptide and making the copy number of β-glucosidase 4 copies.

    These co-presenting strains have higher ethanol productivity than the existing cellulase surface display laboratory yeast MT8-1 / pBG211 / pEG23u31H6 (Murai et al., Applied and Environmental Microbiology, 2002, p.5136-5141). Indicated. This strain is a transformed strain with an autonomously replicating multicopy plasmid. Specifically, a plurality of copies of plasmid pBG211 for surface display of Aspergillus aculeatus β-glucosidase and plasmid pEG23u31H6 for surface display of Trichoderma reesei endoglucanase (EGII) are retained in the plasmid state. The number of copies of the introduced gene is generally considered to be several tens of copies. Each plasmid does not contain the linker used in the present invention.

    The yeast of the present invention (super yeast) produced in this example showed high ethanol productivity although the transgene copy number was smaller than that of the existing strain. The reason is considered to be the result of a synergistic effect due to multiple factors such as improved stability of the traits due to genomic integration, adaptability to host ethanol fermentation, and high expression by the SED1 promoter.

Ethanol-fermented plant material from a model substrate As a model substrate of a cellulose degradation product contained in a treatment solution obtained by a known hydrolysis treatment method, substrate solution A (5 wt% glucose, 2 wt% cellobiose, 20 mM phosphoric acid) Sodium buffer (pH 5.0)) and substrate solution B (5 wt% glucose, 2 wt% cellobiose, 20 mM sodium phosphate buffer (pH 5.0), 2 wt% casamino acid) were used.

To each substrate solution, the parent strain, sake yeast GRI-117-UK strain, and the sake yeast UK / UDB7CBII / KDB7CBII strain prepared as described above were added so that the OD ₆₀₀ was 20, respectively, at 30 ° C. The reaction was performed for 24 hours. Table 1 below shows the ethanol concentration, glucose concentration, and ethanol yield in the bacterial solution after the reaction.

^* Ethanol yield: Ratio of ethanol weight to total sugar weight

    Since the maximum yield when all the substrate sugars are decomposed to ethanol is 0.520, when the substrate solution A is ethanol-fermented using the cellulolytic enzyme-presenting yeast of the present invention, it is very efficient. It can be seen that sugar was alcohol fermented well.

It is a figure which shows the relationship between linker length and enzyme activity. It is a figure which shows the relationship between a secretory signal sequence and enzyme activity. It is a figure which shows an enzyme activity when both linker length and a secretory signal sequence are optimized. FIG. 4 is a diagram showing the rate of ethanol production by the cellulolytic enzyme-presenting yeast of the present invention. FIG. 3 is a graph showing the rate of ethanol production by the cellulolytic enzyme-presenting yeast of the present invention.

Claims (14)

A sake yeast in which the following polynucleotides (a) and (b) are integrated into a chromosome, and β-glucosidase and endoglucanase are co-presented on the surface.
(a) a polynucleotide comprising, in order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence that encodes β-glucosidase, and a sequence that encodes a cell surface localized protein or a cell membrane binding region thereof
(b) a polynucleotide comprising, in order from the 5 ′ end, a secretory signal sequence that functions in yeast cells, a sequence encoding endoglucanase, and a sequence encoding a cell surface localized protein or a cell membrane binding region thereof The polynucleotide of (a) is a linker having a length of 6 to 90 bases between a sequence encoding β-glucosidase and a sequence encoding a cell surface localized protein or a cell membrane binding region thereof,
The polynucleotide of (b) is a linker having a length of 6 to 90 bases between a sequence encoding endoglucanase and a sequence encoding a cell surface localized protein or a cell membrane binding region thereof,
The sake yeast according to claim 1.   The sake yeast according to claim 1 or 2, wherein the secretory signal sequence of (a) is a combination of two or more secretory signal sequences.   The sake yeast according to any one of claims 1 to 3, wherein the secretory signal sequence of (a) is a combination of two or more secretory signal sequences.   The sake yeast according to any one of claims 1 to 4, wherein the secretory signal sequence of (a) is a combination of a secretory signal sequence inherent in secreted β-glucosidase and a secretory signal sequence different from the secretory signal sequence.   The secretory signal sequence of (a) is (1) the secretory signal sequence of Aspergillus oryzae-derived β-glucosidase (BGL7) and the secretory signal sequence of Aspergillus oryzae-derived β-glucosidase (BGL1) The sake yeast according to any one of claims 1 to 5, which is a combination of (2) a secretory signal sequence of BGL7 and a secretory signal sequence of Rhizopus oryzae-derived glucoamylase (GlaR).   The secretion signal sequence of (b) is a combination of the secretion signal sequence originally possessed by endoglucanase and the secretion signal of endoglucanase derived from Aspergillus oryzae (CelA). Sake yeast.   The sake yeast according to any one of claims 1 to 7, wherein the secretion signal sequence of (b) is a combination of the secretion signal sequence of CelA and the secretion signal sequence of endoglucanase (CelB) derived from Aspergillus oryzae. .   The sake yeast according to any one of claims 1 to 8, wherein the nucleotides of (a) and / or (b) are incorporated into a multiple copy chromosome.   The sake yeast according to any one of claims 1 to 9, wherein the sequence encoding the cell surface localized protein or the cell membrane-binding region thereof is a sequence encoding 320 to 600 amino acids from the C-terminal of yeast α-agglutinin. .     Sake yeast in which β-glucosidase and endoglucanase are bound to a cell membrane via a cell surface localized protein or a cell membrane binding region thereof, respectively.   A linker peptide consisting of 2 to 30 amino acid residues is interposed between β-glucosidase and cell surface localized protein or its cell membrane binding region, and 2 between endoglucanase and cell surface localized protein or its cell membrane binding region. The sake yeast according to claim 11, wherein a linker peptide consisting of -30 amino acid residues is interposed.   The manufacturing method of ethanol which decomposes | disassembles the polymer by the beta-glucoside bond of glucose in the presence of the sake yeast in any one of Claims 1-11, and obtains ethanol by fermentation of the produced | generated glucose.   14. The method according to claim 13, wherein the polymer having glucose β-glucoside bonds is contained in a treated product obtained by hydrolyzing the plant material.

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