# Patent

## Title
Polynucleotide for cell surface layer expression

## Abstract

Disclosed is a polynucleotide for cell surface expression. The polynucleotide of the invention comprises a promoter, a secretion signal sequence, a sequence encoding an intended protein, and a sequence encoding a cell surface-localized protein or a cell membrane-binding domain thereof, wherein the promoter is a promoter of a gene encoding the cell surface-localized protein. Provided is a polynucleotide for cell surface expression allowing the production of yeast displaying an enzyme on the cell surface with a high activity.

## Background
The present application is a National Stage Application of PCT/JP2014/058189, filed Mar. 25, 2014, which claims priority from Japanese Patent Application No. 2013-062795, filed Mar. 25, 2013.
TECHNICAL FIELD
The present invention relates to a polynucleotide for cell surface expression, and more specifically relates to a polynucleotide for cell surface expression that allows for the production of a yeast displaying an enzyme on the cell surface with a high activity.
BACKGROUND ART
Recently, since there is a concern that fossil fuels are being exhausted, alternative fuels have been developed. In particular, bioethanol derived from biomass has attracted attention. This is because biomass is a renewable resource, which exists in large amounts on the earth, and can be used without increasing carbon dioxide in the atmosphere (carbon neutral) so as to contribute to the prevention of global warming.
The attempt has been made that fermentation microorganisms that originally cannot utilize main components of soft biomass, such as cellulose or hemicellulose, are modified by bioengineering procedures to attain ethanol fermentation directly from non-edible carbon sources.
As such bioengineering procedures, cell surface engineering are suitably used. Examples of the cell surface engineering include a method using a GPI anchor protein of a cell surface-localized protein. As such a GPI anchor protein, various proteins are known (Patent Documents 1 to 4 and Non-Patent Documents 1 to 5, for example). Meanwhile, a promoter may be involved in the expression of protein in the cell surface engineering. Examples of known promoters exhibiting a high promoter activity in yeast cells include yeast SED1 promoter, GAPDH promoter, PGK1 promoter, ADH1 promoter, and the like (Patent Documents 2 to 4, for example).
PRIOR ART DOCUMENTS
Patent Documents


    Patent Document 1: Japanese Laid-Open Patent Publication No. 2011-160727
    Patent Document 2: Japanese Laid-Open Patent Publication No. 2008-86310
    Patent Document 3: Japanese Laid-Open Patent Publication No. 2007-189909
    Patent Document 4: Japanese Laid-Open Patent Publication No. 2005-245335
    Patent Document 5: Japanese Laid-Open Patent Publication No. 2011-30563


Non-Patent Documents


    Non-Patent Document 1: Biotechnol. Lett., 2010, vol. 32, pp. 1131-1136
    Non-Patent Document 2: Mol. Microbiol., 2004, vol. 52, pp. 1413-1425
    Non-Patent Document 3: Appl. Environmen. Microbiol., 1997, vol. 63, pp. 615-620
    Non-Patent Document 4: Biotechnol. Lett., 2010, vol. 32, pp. 255-260
    Non-Patent Document 5: J. Bacteriol., 1997, vol. 179, pp. 1513-1520
    Non-Patent Document 6: Nature Methods, 2009, vol. 6, pp. 343-345
    Non-Patent Document 7: Yeast, 1998, vol. 14, pp. 115-132
    Non-Patent Document 8: Appl. Microbiol. Biotechnol., 2006, vol. 72, pp. 1136-1143
    Non-Patent Document 9: Appl. Microbiol. Biotechnol., 2010, vol. 85, pp. 1491-1498
    Non-Patent Document 10: Enzyme Microb. Technol., 2012, vol. 50, pp. 343-347


SUMMARY OF INVENTION
Problems to be Solved by the Invention
It is an object of the present invention to provide a polynucleotide for cell surface expression that allows for the production of a yeast displaying an enzyme on the cell surface with a high activity.
Means for Solving the Problems
The present invention provides a polynucleotide for cell surface expression, comprising: a promoter, a secretion signal sequence, a sequence encoding an intended protein, and a sequence encoding a cell surface-localized protein or a cell membrane-binding domain thereof, wherein the promoter is a promoter of a gene encoding the cell surface-localized protein.
In an embodiment, the cell surface-localized protein is SED1 or CWP2.
The present invention also provides a polynucleotide for cell surface expression, comprising: a promoter, a secretion signal sequence, a sequence encoding an intended protein, and a sequence encoding a cell surface-localized protein or a cell membrane-binding domain thereof, wherein the promoter and the sequence encoding the cell surface-localized protein or the cell membrane-binding domain thereof are derived from any one gene of the genes Sed1 and Cwp2.
In an embodiment, both the promoter and the sequence encoding the cell surface-localized protein or the cell membrane-binding domain thereof are derived from the Sed1 gene; or both the promoter and the sequence encoding the cell surface-localized protein or the cell membrane-binding domain thereof are derived from the Cwp2 gene.
The present invention further provides an expression vector, comprising the polynucleotide for cell surface expression.
The present invention further provides a recombinant yeast into which the polynucleotide for cell surface expression or the expression vector has been introduced.
In an embodiment, the recombinant yeast is obtained from a host yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient.
In a further embodiment, the recombinant yeast is obtained from a host yeast in which SED1 and SSD1 are deficient.
In an embodiment, the recombinant yeast displays at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface.
The present invention also provides a method for producing ethanol, comprising:
performing fermentation culture of the recombinant yeast mentioned above.
Effects of Invention
According to the present invention, provided is a polynucleotide for cell surface expression that allows for the production of a yeast displaying a protein such as an enzyme on the cell surface with a high activity. The yeast into which such a polynucleotide has been introduced can express a protein on the cell surface with a high activity.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing the time course of a β-glucosidase activity during culture for the Gap-Agα1 recombinant strain, Gap-Sed1 recombinant strain, Sed1-Agα1 recombinant strain, and Sed1-Sed1 recombinant strain for BGL1 gene.
FIG. 2 is a graph showing an endoglucanase activity after culture for 48 hours for the Gap-Agα1 recombinant strain, Gap-Sed1 recombinant strain, Sed1-Agα1 recombinant strain, and Sed1-Sed1 recombinant strain for EGII gene.
FIG. 3 is a graph showing the time course of a β-glucosidase activity during culture for the Gap-Agα1 recombinant strain, Sed1-Sed1 recombinant strain, and Cwp2-Cwp2 recombinant strain for BGL1 gene.
FIG. 4 is a graph showing the time course of a β-glucosidase activity during culture for the Sed1-Sed1 recombinant strain, Cwp2-Cwp2 recombinant strain, and Sed1-Cwp2 recombinant strain for BGL1 gene.
FIG. 5 is a graph showing the time course of a β-glucosidase activity during culture for the Sed1-Sed1 recombinant BY4741 SED1Δ strain and Sed1-Sed1 recombinant BY4741 strain for BGL1 gene.
FIG. 6 is a graph showing the time course of a β-glucosidase activity during culture for the Sed1-Sed1 recombinant BY4741 SSD1Δ strain and Sed1-Sed1 recombinant BY4741 strain for BGL1 gene.
FIG. 7 is a graph showing the time course of a β-glucosidase activity during culture for the Sed1-Sed1 recombinant BY4741 strain, Sed1-Sed1 recombinant BY4741 SED1Δ strain, Sed1-Sed1 recombinant BY4741 SSD1Δ strain, and Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ for BGL1 gene.
FIG. 8 is a graph showing the time course of the amount of ethanol produced during fermentation culture for the Sed1-Sed1 recombinant strain and Gap-Agα1 recombinant strain for EGII gene, and an empty vector-introduced strain (no cell surface display).
FIG. 9 is a graph showing the time course of α-amylase and glucoamylase activities during culture for the α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain.
FIG. 10 is a graph showing the time course of the amount of ethanol produced from raw starch by the α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain.



MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
A polynucleotide for cell surface expression is a polynucleotide including a promoter, a secretion signal sequence, a sequence encoding an intended protein, and a sequence encoding a cell surface-localized protein or a cell membrane-binding domain thereof.
(Cell Surface-Localized Protein and Cell Membrane-Binding Domain Thereof)
“Cell surface-localized protein” refers to a protein anchored, or attached or adhered to the cell surface to be localized therein. Known examples of the cell surface-localized protein include a protein modified with a lipid which is anchored to the cell membrane through a covalent bond with a component of the membrane. Herein, “the cell surface-localized protein and the cell membrane-binding domain thereof” may be collectively referred to simply as an “anchor”, based on their roles.
Typical examples of the cell surface-localized protein include a GPI (glycosyl phosphatidyl inositol: glycolipid having, as a basic structure, ethanolamine phosphate-6 mannose α-1,2 mannose α-1,6 mannose α-1,4 glucosamine α-1,6 inositol phospholipid) anchor protein. The GPI anchor protein, which has a glycolipid GPI at its C-terminus, is bound to the surface of a cell membrane through a covalent bond of the GPI with PI (phosphatidyl inositol) in the cell membrane.
The bonding of GPI to the C-terminus of the GPI anchor protein is made as follows. After transcription and translation, the GPI anchor protein is secreted into the lumen of the endoplasmic reticulum by the action of a secretion signal present on the N-terminal side. There is a domain at or near the C-terminus of a GPI anchor protein, and the domain is recognized by a GPI anchor on its binding to the GPI anchor protein, and is called a GPI anchor attachment signal. In the lumen of the endoplasmic reticulum and the Golgi body, the GPI anchor attachment signal domain is cleaved to newly generate C-terminus, and a GPI is bound to the newly generated C-terminus.
The protein bound by the GPI is transported to the cell membrane through secretion vesicles, and is anchored to the cell membrane through a covalent bond of the GPI with PI in the cell membrane. Then, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), and is incorporated into the cell wall to display the protein on the cell surface in the state of anchoring to the cell wall.
In the present invention, it is possible to use a polynucleotide encoding a GPI anchor protein which is a cell surface-localized protein, at its entirety or region including a GPI anchor attachment signal domain which is a cell membrane-binding domain thereof. The cell membrane-binding domain (GPI anchor attachment signal domain) is typically a C-terminal region of the cell surface-localized protein. The cell membrane-binding domain may be any region, as long as it includes the GPI anchor attachment signal domain, and may further include any other portion of the GPI anchor protein, as long as it does not inhibit the enzyme activity of the fusion protein.
The GPI anchor protein may be any protein as long as it functions in a yeast cell. Examples of the GPI anchor protein include α- or α-agglutinin (AGα1, AGA1), TIP1, FLO1, SED1, CWP1, and CWP2, and SED1 and CWP2 are preferred.
SED1 is a main cell surface-localized protein in the stationary phase of yeast Saccharomyces cerevisiae, which is considered to be induced by stress and contribute to maintaining the integrity of the cell wall. The gene, Sed1, for SED1 can be obtained, for example, using a method commonly used by those skilled in the art, based on the sequence information registered in GenBank (GenBank accession number NM_001180385; NCBI Gene ID: 851649). The base sequence of the protein coding region of Sed1 is shown in SEQ. ID. No. 1, and the amino acid sequence of the encoded protein is shown in SEQ. ID. No. 2. The cell membrane-binding domain (GPI anchor attachment signal domain) of SED1 is, for example, a region including positions 110 to 338 of SEQ. ID. No. 2. The polynucleotide encoding SED1 may be a polynucleotide encoding a full-length amino acid sequence of SEQ. ID. No. 2, or may be a polynucleotide encoding a partial sequence (e.g., sequence including the amino acid sequence at positions 110 to 338 of SEQ. ID. No. 2), as long as the anchor function is not impaired.
CWP2 is a cell surface-localized protein of yeast Saccharomyces cerevisiae. The gene, Cwp2, for CWP2 can be obtained, for example, using a method commonly used by those skilled in the art, based on the sequence information registered in GenBank (GenBank accession number NM_001180025; NCBI Gene ID: 853765). The base sequence of the protein coding region of Cwp2 is shown in SEQ. ID. No. 3, and the amino acid sequence of the encoded protein is shown in SEQ. ID. No. 4. The cell membrane-binding domain (GPI anchor attachment signal domain) of CWP2 is, for example, a region including positions 26 to 92 of SEQ. ID. No. 4. The polynucleotide encoding CWP2 may be a polynucleotide encoding a full-length amino acid sequence of SEQ. ID. No. 4, or may be a polynucleotide encoding a partial sequence (e.g., sequence including the amino acid sequence at positions 26 to 92 of SEQ. ID. No. 4), as long as the anchor function is not impaired.
Herein, the polynucleotide may be a polynucleotide encoding a protein composed of an amino acid sequence in which one or several amino acids are deleted from, substituted with, or added to those in a disclosed amino acid sequence, and substantially having functions or effects desired in the present invention. The number of types of mutations (e.g., deletion, substitution, or addition) of amino acids in the disclosed amino acid sequence may be one, or may be two or more in combination. Furthermore, without any particular limitation, the total number of mutations is, for example, 1 or more and 10 or less, or 1 or more and 5 or less. The amino acid substitution may be any substitution, as long as the functions or effects are substantially maintained, but examples thereof include conservative substitution. The conservative substitution may be specifically substitution in the following groups (i.e., substitution between amino acids shown in each parenthesis): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine), or (phenylalanine, tyrosine).
In another embodiment, the polynucleotide may be a polynucleotide encoding a protein having an amino acid sequence having a sequence identity of, for example, 70% or more, with a disclosed amino acid sequence, and substantially having functions or effects desired in the present invention. The sequence identity in the amino acid sequence may be 74% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more.
In this specification, the identity or the similarity of sequences is, as known in the art, a relationship between two or more proteins or between two or more polynucleotides determined by comparing the sequences. The “identity” of sequences refers to the degree to what extent protein or polynucleotide sequences are identical to each other, as determined from alignments of the protein or polynucleotide sequences, or, in some cases, from alignments of continuous partial sequences. Furthermore, the “similarity” refers to the degree to what extent protein or polynucleotide sequences are similar to each other, as determined from alignments of the protein or polynucleotide sequences, or, in some cases, from alignments of continuous partial sequences. More specifically, it is determined from the identity and the conservation of sequences (substitution maintaining specific amino acids in the sequences or physicochemical characteristics in the sequences). Note that the similarity is referred to as “Similarity” in a sequence homology search result of BLAST (described later). The method for determining the identity and the similarity is preferably a method designed such that alignments are formed as long as possible between sequences that are to be compared. The method for determining the identity and the similarity is provided as a publicly available program. The identity and the similarity can be determined, for example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (e.g., Altschul et at, J. Mol. Biol., 1990, 215: 403-410; Altschul et al., Nucleic Acids Res., 1997, 25: 3389-3402). There is no particular limitation on the conditions when using software such as the BLAST, but it is preferable to use default values.
In another embodiment, the polynucleotide may be a polynucleotide that hybridizes under stringent conditions with a DNA composed of a base sequence that is complementary to a DNA composed of a disclosed base sequence. The stringent conditions are, for example, a condition under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, it is a condition under which a nucleic acid having a high base sequence identity, that is, a complementary strand of a DNA composed of a base sequence having an identity of, for example, 65% or more, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more, with a disclosed base sequence hybridizes, and a complementary strand of a nucleic acid having a homology lower than that does not hybridize. More specifically, it is a condition under which the sodium salt concentration is, for example, 15 to 750 mM, 50 to 750 mM, or 300 to 750 mM, the temperature is, for example, 25 to 70° C., 50 to 70° C., or 55 to 65° C., and the formamide concentration is, for example, 0 to 50%, 20 to 50%, or 35 to 45%. Furthermore, under the stringent conditions, the filter washing condition after the hybridization is a condition under which the sodium salt concentration is, for example, 15 to 600 mM, 50 to 600 mM, or 300 to 600 mM, and the temperature is, for example, 50 to 70° C., 55 to 70° C., or 60 to 65° C. The hybridization can be performed using a well known method such as the method described in Sambrook et at, Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory (2001). As the temperature is higher or the salt concentration is lower, the stringency increases, and thus, a polynucleotide having a higher homology can be isolated.
In another embodiment, the polynucleotide may be a polynucleotide having a base sequence having an identity of, for example, 65% or more, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more, with a disclosed base sequence, and substantially having the functions or effects desired therefor.
For example, in a sequence having: secretion signal sequence-structural gene encoding cell surface-localized protein-sequence encoding GPI anchor attachment recognition signal, the entire or a partial sequence of the structural gene encoding the cell surface-localized protein can be substituted with a sequence encoding an intended protein.
The polynucleotide (gene) encoding the cell surface-localized protein or the cell membrane-binding domain thereof may be obtained from microorganisms having them, for example, by PCR with primers or hybridization with a probe, based on known sequence information. The polynucleotide can be also used by excising it from an existing vector containing the same. Alternatively, the polynucleotide may be synthesized as a nucleic acid fragment using various nucleic acid sequence synthesis methods known in the art, such as chemical synthesis.
(Secretion Signal Sequence)
The “secretion signal sequence” is a polynucleotide sequence encoding a secretion signal peptide.
The secretion signal peptide is a peptide typically linked to the N-terminus of a secretory protein which is secreted out of the cell containing periplasm, and the peptide is similar in structure among organisms, and is, for example, composed of about 20 amino acids, including a sequence of basic amino acids near the N-terminus, followed by rich in hydrophobic amino acids. The secretion signal is typically removed through the degradation by a signal peptidase when the secretory protein is secreted from inside the cell through the cell membrane into the outside of the cell.
In the present invention, any polynucleotide sequence encoding a secretion signal peptide capable of secreting the intended protein out of a yeast cell can be used, and there is no limitation on the origin thereof. Preferable examples thereof include sequences encoding secretion signal peptide of glucoamylase of Rhizopus oryzae and the like, secretion signal peptide of glucoamylase of Aspergillus oryzae, secretion signal peptide of α- or α-agglutinin of yeast Saccharomyces cerevisiae, secretion signal peptide of a factor of yeast Saccharomyces cerevisiae, and the like. In particular, in view of secretion efficiency, it is preferable to use the secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase. Also, it is preferable to use the secretion signal peptide sequence of Aspergillus oryzae-derived glucoamylase. It is also possible to use a sequence encoding secretion signal peptide originally contained in the intended protein.
(Promoter)
The promoter may be any promoter, as long as it has a promoter activity. The “promoter activity” refers to an activity that allows a transcription factor to be bound to a promoter region, thereby inducing transcription. The promoter may be cut out with a restriction enzyme from, for example, microorganisms or phages having a desired promoter region. The DNA fragment of a promoter region can be obtained by amplifying a desired promoter region by PCR with primers provided with a restriction enzyme recognition site or a site overlapping a cloning vector as necessary. It is also possible to chemically synthesize a desired promoter based on base sequence information of an already known promoter region.
The promoter contained in the polynucleotide for cell surface expression is preferably a promoter of a gene encoding the cell surface-localized protein or the cell membrane-binding domain thereof used as an anchor, or a promoter contained natively in the gene. That is to say, the promoter and the anchor (the cell surface-localized protein or the cell membrane-binding domain thereof) are preferably derived from the same gene.
According to the invention, the promoter used is preferably Sed1 and Cwp2 promoters. The base sequences of the Sed1 and Cwp2 promoters are respectively shown, for example, in Saccharomyces cerevisiae S288c chromosome 1V, complete sequence (GenBank accession number NC 001136) and Saccharomyces cerevisiae S288c chromosome XI, complete sequence (GenBank accession number NC_001143) of GenBank. The base sequence of the Sed1 and Cwp2 promoters are respectively shown in SEQ. ID. Nos. 5 and 6.
For example, when the cell surface-localized protein is SED1, it is preferable that the coding region of the gene Sed1 therefor is used as a sequence encoding the cell surface-localized protein and the promoter of the gene Sed1 is used as a promoter, and when the cell surface-localized protein is CWP2, it is preferable that the coding region of the gene Cwp2 therefor is used as a sequence encoding the cell surface-localized protein and the promoter of the gene Cwp2 is used as a promoter (in these cases, the promoter and the anchor are derived from the same gene). Meanwhile, the coding region of the gene Cwp2 may be used with the promoter of Sed1, and the coding region of the gene Sed1 may be used with the promoter of Cwp2. The coding regions and the promoters described above may have a base sequence in which one or two or more (e.g., several) nucleotides are mutated (e.g., deleted, added, or substituted) from those in their original base sequence as described above, as long as the intended functions are realized.
(Sequence Encoding Intended Protein)
There is no particular limitation on the type or origin of the intended protein. Examples of the type of the intended protein include enzyme, antibody, ligand, fluorescent protein, and the like. Examples of the enzyme include cellulose-degrading enzyme, starch-degrading enzyme, glycogen-degrading enzyme, xylan-degrading enzyme, chitin-degrading enzyme, lipid-degrading enzyme, and the like, and more specifically include endoglucanase, cellobiohydrolase, β-glucosidase, amylase (e.g., glucoamylase and α-amylase), lipase, and the like.
The polynucleotide sequence encoding the intended protein is preferably a cDNA sequence with no intron.
The polynucleotide sequence encoding the intended protein may be a sequence encoding the full-length, or may be a sequence encoding a partial region of the intended protein, as long as the activity of the intended protein is exhibited. Furthermore, as described above, it may be a base sequence in which one or two or more (e.g., several) nucleotides are mutated (e.g., deleted, added, or substituted), or a base sequence encoding a protein composed of an amino acid sequence in which one or two or more (e.g., several) amino acids are mutated (e.g., deleted, added, or substituted), from those in a base sequence encoding a native protein, as long as the activity of the intended protein is exhibited.
The polynucleotide (gene) encoding the intended protein may be obtained from enzyme-producing microorganisms, for example, by PCR with primers or hybridization with a probe, based on known sequence information. Furthermore, the polynucleotide can be used by excising it, preferably in the form of an expression cassette, from an existing vector containing the polynucleotide encoding the intended protein.
Hereinafter, a description will be given using cellulose-degrading enzymes and starch-degrading enzymes as examples of the intended protein.
The cellulose-degrading enzyme refers to any enzyme that can cleave a β1,4-glycosidic bond. It may be derived from any microorganisms that produce a cellulose-hydrolyzing enzyme. Typical examples of the microorganisms that produce a cellulose-hydrolyzing enzyme include microorganisms belonging to the genus Aspergillus (e.g., Aspergillus aculeatus, Aspergillus niger, and Aspergillus oryzae), the genus Trichoderma (e.g., Trichoderma reesei), the genus Clostridium (e.g., Clostridium thermocellum), the genus Cellulomonas (e.g., Cellulomonas fimi and Cellulomonas uda), the genus Pseudomonas (e.g., Pseudomonas fluorescence), and the like.
Hereinafter, endoglucanase, cellobiohydrolase, and β-glucosidase will be described as typical cellulose-degrading enzymes, but the cellulose-degrading enzyme is not limited thereto.
Endoglucanase is an enzyme that is usually referred to as cellulase, and it cleaves cellulose from the inside of the molecule to generate glucose, cellobiose, and cello-oligosaccharide (“cellulose molecule inside cleaving”). There are five types of endoglucanase and they are respectively referred to as endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase I\ and endoglucanase V. They are different from each other in terms of amino acid sequences, but commonly have the action of cellulose molecule inside cleaving. For example, Trichoderma reesei-derived endoglucanase (especially endoglucanase II: EGII (Patent Document 5, for example)) may be used, but there is no limitation thereto.
Cellobiohydrolase degrades cellulose from either the reducing terminus or the non-reducing terminus thereof to liberate cellobiose (“cellulose molecule terminal cleaving”). There are two types of cellobiohydrolase and they are respectively referred to as cellobiohydrolase I and cellobiohydrolase II. They are different from each other in terms of amino acid sequences, but commonly have the action of cellulose molecule terminal cleaving. For example, Trichoderma reesei-derived cellobiohydrolase (especially cellobiohydrolase II: CBHII (Patent Document 5, for example)) may be used, but there is no limitation thereto.
β-Glucosidase is an exo-type hydrolytic enzyme that liberates glucose units from the non-reducing terminus of cellulose (“glucose unit cleaving”). β-Glucosidase can cleave a β1,4-glycosidic bond between aglycone or a sugar chain and β-D-glucose, and hydrolyze cellobiose or cello-oligosaccharide, to generate glucose. β-Glucosidase is a typical example of an enzyme that can hydrolyze cellobiose or cello-oligosaccharide. Currently, there is one type of known β-glucosidase and it is referred to as β-glucosidase 1. For example, Aspergillus aculeatus-derived β-glucosidase (especially β-glucosidase 1: BGL1 (Non-Patent Document 8, for example)) may be used, but there is no limitation thereto.
For favorable cellulose hydrolysis, it is preferable to combine enzymes that hydrolyze cellulose in different ways. Various enzymes that hydrolyze cellulose in different ways, such as cellulose molecule inside cleaving, cellulose molecule terminal cleaving, and glucose unit cleaving, may be combined as appropriate. Examples of the enzymes that have the respective ways of hydrolysis include, but are not limited to, endoglucanase, cellobiohydrolase, and β-glucosidase. A combination of enzymes that hydrolyze cellulose in different ways may be selected from the group consisting of, for example, endoglucanase, cellobiohydrolase, and β-glucosidase. Since it is desirable that glucose, which is a constituent sugar of cellulose, is eventually produced, at least one enzyme that can generate glucose is preferably included. Examples of the enzyme that can generate glucose include endoglucanase, in addition to glucose unit cleaving enzyme (e.g., β-glucosidase). For example, β-glucosidase, endoglucanase, and cellobiohydrolase may be displayed on the cell surface in a yeast.
Hereinafter, glucoamylase and α-amylase will be described as typical starch-degrading enzymes, but the starch-degrading enzyme is not limited thereto.
Glucoamylase is officially referred to as glucan 1,4-α-glucosidase, and is also referred to as 1,4-α-D-glucan glucohydrolase, exo 1,4-α-glucosidase, γ-amylase, lysosomal α-glucosidase, or amyloglucosidase. Glucoamylase hydrolyzes an α-1,4-bond at the non-reducing terminus of a sugar chain in an exo manner, to produce one molecule of dextrose. There is also known glucoamylase that cleaves an α-1,6-bond. Examples of the glucoamylase include, but are not limited to, Rhizopus oryzae-derived glucoamylase (Non-Patent Document 9, for example).
α-Amylase is also referred to as 1,4-α-D-glucan glucanohydrolase, or glycogenase, and is an enzyme irregularly cleaves an α-1,4-bond of starch or glycogen to produce polysaccharide, maltose, or oligosaccharide. It is a general enzyme widely distributed in animals, plants, and microorganisms. Examples of the α-amylase include, but are not limited to, Streptococcus bovis-derived α-amylase (Non-Patent Document 10, for example)).
For favorable starch hydrolysis, it is preferable to combine enzymes that hydrolyze starch in different ways. In an embodiment, both α-amylase and glucoamylase can be displayed on the cell surface in a yeast.
(Terminator)
The polynucleotide for cell surface expression may further include a terminator.
The terminator may be any terminator, as long as it has a terminator activity. The “terminator activity” refers to an activity that terminates transcription in a terminator region. The terminator may be any terminator, as long as it has a terminator activity, and may be cut out with a restriction enzyme from, for example, microorganisms or phages having a desired terminator region. The DNA fragment of a terminator region can be obtained by amplifying a desired terminator region by PCR with primers provided with a restriction enzyme recognition site or a site overlapping a cloning vector as necessary. It is also possible to chemically synthesize a desired terminator based on base sequence information of an already known terminator region.
Examples of the terminator include α-agglutinin terminator, ADH1 (aldehyde dehydrogenase) terminator, GAPDH (glyceraldehyde-3′-phosphate dehydrogenase) terminator, and the like.
(Construction of Polynucleotide for Cell Surface Expression)
A sequence encoding the anchor (the cell surface-localized protein or the cell membrane-binding domain thereof) is ligated to a sequence encoding the intended protein (structural gene), together with the secretion signal sequence in a desired arrangement, and the ligate is arranged downstream of the promoter. These sequences are arranged in the polynucleotide for cell surface expression such that, for example, the intended protein is linked to the N-terminus of the anchor (the cell surface-localized protein or the cell membrane-binding domain thereof) when the intended protein is expressed. That is to say, the sequence encoding the intended protein is positioned on the 5′ side of the sequence encoding the anchor. Furthermore, the sequence encoding the intended protein is arranged downstream of the secretion signal sequence.
The terminator can be arranged downstream of the ligate of the above-described factors (the promoter, the secretion signal sequence, the sequence encoding the intended protein, and the sequence encoding the anchor).
The synthesis and the binding of DNAs having the various sequences described above may be performed using a method commonly used by those skilled in the art. For example, the binding of the secretion signal sequence and the structural gene of the intended protein can be carried out by way of site specific mutation or one-step isothermal assembly (Non-Patent Document 6). Such a procedure allows for accurately cleaving a secretion signal sequence and expressing an active enzyme.
Also, the region (structural gene) encoding the intended protein (e.g., endoglucanase, cellobiohydrolase, or β-glucosidase), the secretion signal, and the expression regulatory sequences such as the promoter and the terminator can be excised as appropriate in a form suitable for vector preparation from a known plasmid containing them, and an insert can be prepared together with the sequence encoding the cell surface-localized protein or the cell membrane-binding domain thereof.
(Expression Vector)
The expression vector may be a plasmid vector, or may be an artificial chromosome. If a yeast is used as a host, the vector is preferably in the form of a plasmid because a vector can be easily prepared and a yeast cell can be easily transformed. In order to simplify the procedure for obtaining a DNA, the vector is preferably a yeast—Escherichia coli shuttle vector. As necessary, the vector may contain regulatory sequences (operator, enhancer, etc.). Such a vector has, for example, a replication origin (Ori) of a 2 μm plasmid of a yeast and a replication origin of ColE1, as well as a yeast selectable marker (described below) and an Escherichia coli selectable marker (drug-resistant gene, etc.).
As the yeast selectable marker, any known markers may be used. Examples thereof include drug-resistant genes, and auxotrophic marker genes (e.g., a gene encoding imidazoleglycerol-phosphate dehydrogenase (HIS3), a gene encoding beta-isopropyl-malate dehydrogenase (LEU2), a gene encoding tryptophan synthase (TRP5), a gene encoding argininosuccinate lyase (ARG4), a gene encoding N-(5′-phosphoribosyl) anthranilate isomerase (TRP1), a gene encoding histidinol dehydrogenase (HIS4), a gene encoding orotidine-5-phosphate decarboxylase (URA3), a gene encoding dihydroorotate dehydrogenase (URA1), a gene encoding galactokinase (GAL1), a gene encoding alpha-aminoadipate reductase (LYS2), etc.). For example, auxotrophic marker genes (e.g., HIS3, LEU2, URA1, TRP1 deficient markers, etc.) may be preferably used.
(Yeast)
There is no particular limitation on the yeast that is used as a host, as long as it belongs to Ascomycetous yeasts. Of these, yeasts belonging to Saccharomycetaceae are preferable, and yeasts belonging to Saccharomyces are more preferable.
The yeast of the present invention is obtained by introducing the polynucleotide or the expression vector of the present invention to a host yeast. To “introduce” include not only to introduce a gene intended to be expressed in the polynucleotide or the expression vector into a host cell but also to allow it to be expressed in the host cell. There is no particular limitation on the procedure of introducing, and known procedures may be used. Typical examples thereof include transforming a yeast with the expression vector of the present invention as described above. There is no particular limitation on the procedure of transformation, and known procedures, such as transfection such as calcium phosphate treatment, electroporation, lipofection, DEAE dextran treatment, lithium acetate treatment, or protoplast, or microinjection may be used without limit. The introduced gene may be present in the form of a plasmid, or may be present in the form inserted into a yeast chromosome or in the form incorporated into a yeast chromosome by homologous recombination.
The yeast into which the polynucleotide of the present invention has been introduced can be selected according to a common procedure, using, as an indicator, for example, the character by the yeast selectable marker or the activity of the intended protein.
Furthermore, it is possible to see that the intended protein is anchored to the cell surface (displayed on the cell surface) of the obtained yeast according to a common procedure. Examples thereof include action of an antibody against this protein and a fluorescence-labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase on a yeast subject; reaction of an antibody against this protein and a biotin-labeled secondary antibody with a yeast subject, and then a fluorescence-labeled streptavidin; and the like.
It is also possible to transform a yeast so as to express multiproteins on the cell surface. In this case, a plurality of expression vectors containing respective gene expression cassettes for sequences encoding multiproteins may be constructed, or a plurality of gene expression cassettes may be placed in one expression vector. For example, δ integration may be used (Patent Document 5).
In an embodiment, for example, a yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient is used as a host for transformation. The host is preferably a SED1 deficient yeast, and more preferably a SED1 and SSD1 double-deficient yeast. The SED1 is as described above. The SSD1 is a negative regulator to a yeast stress responsive gene, such as SED1. The NCBI gene registration number for a gene for SSD1 is NCBI Gene ID: 851887 (Genbank accession number NM_001180601.1). The base sequence of the gene for SSD1 derived from Saccharomyces cerevisiae and the encoded amino acid sequence thereby are respectively shown in SEQ. ID. Nos. 38 and 39.
The yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient can be obtained, for example, by disrupting, or suppressing expression of, genes encoding these proteins in the host yeast. Examples of such “deficiency” include suppressing the amount of normal protein produced, and producing or facilitating dysfunctional mutant proteins. Examples of the gene manipulation therefor include transgenesis, gene knockout, and knockin. In an embodiment, for example, the SED1 and SSD1 double-deficient yeast is a double-disrupted strain in which both the genes for SED1 and SSD1 are disrupted.
Such deficient yeasts may be produced by preparing primers and the like as appropriate based on known gene sequence information and performing the gene manipulation as mentioned above or may be commercially available deficient strains. Examples of such commercially available deficient strains include Yeast Knockout Collection (available from Open Biosystems) for yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain), and the like.
(Method for Producing Ethanol)
The yeast displaying at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface according to the present invention can be used for ethanol production. In an embodiment, it is a yeast displaying at least one enzyme selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase on the cell surface (alternatively referred to as “cellulase-displaying yeast” herein). Such a yeast may be a yeast displaying two types of enzymes selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase; or endoglucanase, cellobiohydrolase, and β-glucosidase on the cell surface. In another embodiment, it is a yeast displaying α-amylase and/or glucoamylase on the cell surface (alternatively referred to as “amylase-displaying yeast” herein). The yeast may display both a cellulose-degrading enzyme and a starch-degrading enzyme on the cell surface. It is preferable to use a cassette containing the coding region of the gene Sed1 and the promoter of the gene Sed1, as a cell surface expression cassette.
The cellulase-displaying yeast may use cellulose and a saccharified product thereof, as a fermentation substrate. The cellulase-displaying yeast may use starch and a saccharified product thereof, as a fermentation substrate. Examples of the obtaining source or the material of the fermentation substrate containing cellulose and a saccharified product thereof and the fermentation substrate containing starch and a saccharified product thereof include biomass. The biomass refers to industrial resources that are not exhaustible resources and are derived from biological materials of recently living organisms. That is to say, biomass refers to renewable organic resources derived from organism, excluding fossil resources. Biomass may include cellulose and/or starch. There is no particular limitation on the biomass, and examples thereof include resource crops and wastes thereof. There is no particular limitation on the resource crops, and examples thereof include corn and sugarcane. Examples of the wastes of resource crops include wastes generated when processing these resource crops. The use of lignocellulosic biomass is preferable because it does not compete with food. There is no particular limitation on the lignocellulosic biomass, and examples thereof include portions (e.g., chaff, roots, stems, and leaves) excluding edible portions of Poaceae plants such as Oryza sativa, wheat, Miscanthus sinensis; Phragmites communis, and the like, and wastes generated from product of these portions.
As necessary, before fermentation, the fermentation substrate material (e.g., biomass) may be subjected to pre-treatment. With such pre-treatment, polysaccharide (e.g., cellulose and/or starch) in biomass can be degraded through “saccharification” into oligosaccharide or monosaccharide. There is no particular limitation on the pre-treatment process, but examples thereof include enzymatic process, diluted sulfuric acid process, and hydrothermal decomposition process. In view of the cost, diluted sulfuric acid process and hydrothermal process are preferable. In diluted sulfuric acid process, for example, the material is treated with 1 to 5% (v/v) of diluted sulfuric acid at 180 to 200° C. for about 5 minutes to 1 hours. In hydrothermal process, for example, the material is treated with water at 130 to 300° C. at about 10 MPa.
The cell surface displaying yeast can be cultured under aerobic conditions before fermentation, to increase the number of cells. The recombinant yeast can be cultured as appropriate using a method well known to those skilled in the art. The pH of the medium is, for example, 4 to 6, and preferably 5. During aerobic culture, the dissolved oxygen concentration in the medium is, for example, 0.5 to 6 ppm, preferably 1 to 4 ppm, and more preferably 2 ppm. The culture temperature is, for example, 20 to 45° C., preferably 25 to 35° C., and more preferably 30° C. The culture is preferably performed until the number of yeast cells is, for example, 10 g (wet weight)/L or more, preferably 12.5 g (wet weight)/L, and more preferably 15 g (wet weight)/L or more, and the culture time is, for example, about 24 to 96 hours.
During fermentation culture, culturing conditions typically applied to yeasts may be selected and used as appropriate. Typically, in culture for fermentation, stationary culture, shaking culture, aerated and stirred culture, or the like may be used. The aeration condition can be selected as appropriate from anaerobic condition, microaerobic condition, aerobic condition, and the like. The culture temperature is, for example, 25 to 45° C., preferably 30 to 40° C., and more preferably 35° C. The culture time may be set to any time as necessary, and may be, for example, in a range of 24 to 120 hours. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. The fermentation medium may contain medium components that may be added for culturing yeasts, as necessary, in addition to the fermentation substrate.
After the end of the ethanol fermentation, a process of collecting an ethanol-containing fraction from the culture solution (fermentation liquor) and also a process of purifying or concentrating the fraction may be performed. These processes and means necessary therefor are selected as appropriate by those skilled in the art.
EXAMPLES
Hereinafter, the present invention will be described by way of examples, but the present invention is not limited thereto.
The yeast Saccharomyces cerevisiae BY4741 strain (Non-Patent Document 7) used in the examples was obtained from Invitrogen.
All PCR procedures shown in the examples were performed using KOD-Plus-Neo-DNA polymerase (manufactured by Toyobo Co., Ltd.).
All gene introductions into yeasts shown in the examples were performed by lithium acetate treatment procedure.
Example 1: Reparation of Vector Containing Expression Cassette for Cell Surface Display and Vector for Cellulase-Displaying
A DNA fragment containing the coding region of Saccharomyces cerevisiae-derived cell surface-localized protein gene Sed1 (hereinafter, alternatively referred to as “SED1” for the sake of convenience) was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair (SED1α-XhoI-F (SEQ. ID. No. 7) and SED1α-BsrGI-R (SEQ. ID. No. 8)). This fragment was treated with XhoI and BsrGI, and was ligated to a vector plasmid pIBG13 (vector for cell surface expression (Non-Patent Document 8) having an auxotrophic marker gene HIS3 and a BGL1 expression cassette (i.e., cassette in which a GAPDH (glyceraldehyde-3′-phosphate dehydrogenase) promoter, a secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase, the coding region of BGL1, a 3′ half region of α-agglutinin gene (a region of nucleotide positions 991 to 1953 in the coding region of α-agglutinin gene), and a terminator region 445 bp downstream of the coding region were arranged in this order) treated in a similar manner. The obtained plasmid was named pIBG13S.
A DNA fragment containing the promoter region of Saccharomyces cerevisiae-derived cell surface-localized protein gene Sed1 was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair SED1p-CBA-F (SEQ. ID. No. 9) and SED1p-CBA-R (SEQ. ID. No. 10). This fragment was ligated, by one-step isothermal assembly, to a fragment amplified using a vector plasmid pIBG13 as a template with a primer pair pIBGvsp-CBA-F (SEQ. ID. No. 11) and pIBGvsp-CBA-R (SEQ. ID. No. 12). The obtained plasmid was named pISpBG13.
The preparation of Aspergillus aculeatus-derived β-glucosidase 1 (BGL1) gene, Trichoderma reesei-derived endoglucanase II (EGII) gene, and Trichoderma reesei-derived cellobiohydrolase II (CBHII) gene were carried out as set forth below.
Gene fragments of BGL1, EGII, and CBHII genes were prepared by PCR, respectively using pIBG13, pδU-PGAGEGII (a vector for cell surface expression of EGII having a 3′ half region of α-agglutinin gene: Patent Document 5), and pδU-PGAGCGHII (a vector for cell surface expression of cellobiohydrolase II having a 3′ half region of α-agglutinin gene: Patent Document 5) as templates, with a primer pair (BGL1-NcoI-F (SEQ. ID. No. 13) and BGL1-PG-R (SEQ. ID. No. 14)) for BGL1, a primer pair (EGII-NcoI-F (SEQ. ID. No. 15) and EGII-XhoI-R (SEQ. ID. No. 16)) for EGII, and a primer pair (CBHII-CBA-F (SEQ. ID. No. 17) and CBHIIaa-CBA-R (SEQ. ID. No. 18) (for Agα1 linkage) or CBHII-CBA-F (SEQ. ID. No. 17) and CBHIIsa-CBA-R (SEQ. ID. No. 19) (for SED1 linkage)) for CBHII.
A DNA fragment containing a 3′ half region of the coding region of Agα1 gene and a DNA fragment containing the coding region of SED1 gene were prepared through amplification by PCR, respectively using pIBG13 and a genome of Saccharomyces cerevisiae BY4741 strain as templates, with a primer pair (AGα1a-PG-F (SEQ. ID. No. 20) and AGα1a-BsrGI-R (SEQ. ID. No. 21)) for Agα1 and a primer pair (SED1α-PG-F (SEQ. ID. No. 22) and SED1α-BsrGI-R (SEQ. ID. No. 8)) for SED1.
DNA fragments (BGL1-Agα1 and BGL1-SED1) in which the DNA fragment containing a 3′ half region of the coding region of Aged gene or the DNA fragment containing the coding region of SED1 gene was ligated in-frame downstream of the BGL1 gene fragment were prepared through amplification by PCR using a mixture of the BGL1 gene fragment, and the DNA fragment containing a 3′ half region of the coding region of Agα1 gene or the DNA fragment containing the coding region of SED1 gene as a template, with a primer pair (BGL1-NcoI-F (SEQ. ID. No. 13) and AGα1a-BsrGI-R (SEQ. ID. No. 21)) for BGL1-Agα1 and a primer pair (BGL1-NcoI-F (SEQ. ID. No. 13) and SED1α-BsrGI-R (SEQ. ID. No. 8)) for BGL1-SED1.
The BGL1-Agα1 and BGL1-SED1 fragments were treated with NcoI and BsrGI, and were ligated in-frame downstream of the secretion signal in a plasmid pIBG13 treated in a similar manner. The obtained plasmids were respectively named pIBG-PG-Agα1 and pIBG-PG-Sed1.
The EGII gene fragment was treated with NcoI and XhoI, and was ligated in-frame downstream of the secretion signal in a plasmid pIBG13 or pIBG13S treated in a similar manner. The obtained plasmids were respectively named pIEG-Agα1 and pIEG-Sed1.
The CBHII gene fragment for Agα1 linkage or the CBHII gene fragment for SED1 linkage was ligated, by one-step isothermal assembly, to a fragment amplified respectively using a vector plasmid pIBG13 or pIBG13S as a template with a primer pair AGα1acb-CBA-F (SEQ. ID. No. 23) and pIBGscb-CBA-R (SEQ. ID. No. 24), or SED1acb-CBA-F (SEQ. ID. No. 25) and pIBGscb-CBA-R (SEQ. ID. No. 24). The obtained plasmids were named pICB-Agα1 and pICB-Sed1.
BGL1-Agα1, BGL1-SED1, EGII-Agα1, and EGII-Sed1 fragments were prepared by respectively treating pIBG-PG-Agα1, pIBG-PG-Sed1, pIEG-Agα1, and pIEG-Sed1 with NcoI and BsrGI, and were ligated in-frame downstream of the secretion signal in a plasmid pISpBG13 treated in a similar manner. The obtained plasmids were respectively named pISpBG-PG-Agα1, pISpBG-PG-Sed1, pISpEG-Agα1, and pISpEG-Sed1.
A DNA fragment containing the promoter region of SED1 gene was prepared through amplification by PCR using pISpBG13 as a template with a primer pair SED1p-CBA-F (SEQ. ID. No. 9) and SED1p-CBA-R (SEQ. ID. No. 10). This fragment was ligated, by one-step isothermal assembly, to a fragment amplified using a plasmid pICB-Sed1 as a template with a primer pair pIBGvsp-CBA-F (SEQ. ID. No. 11) and pIBGvsp-CBA-R (SEQ. ID. No. 12). The obtained plasmid was named pISpCB-Sed1.
A DNA fragment containing a non-coding region on the genome of Saccharomyces cerevisiae was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair NCRv-CBA-F (SEQ. ID. No. 26) and NCRleu2-CBA-R (SEQ. ID. No. 27).
A DNA fragment containing the Saccharomyces cerevisiae-derived Leu2 gene was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair LEU2nc-CBA-F (SEQ. ID. No. 28) and LEU2v-CBA-R (SEQ. ID. No. 29).
The DNA fragment containing the non-coding region and the DNA fragment containing the Leu2 gene were ligated, by one-step isothermal assembly, to a fragment amplified using a plasmid pICB-Agα1 as a template with a primer pair pIBGvleu2-CBA-F (SEQ. ID. No. 30) and pIBGvncr-CBA-R (SEQ. ID. No. 31). The obtained plasmid was named pINCCB-Agα1.
The DNA fragment containing the non-coding region and the DNA fragment containing the Leu2 gene were ligated, by one-step isothermal assembly, to a fragment amplified using a plasmid pISpCB-Sed1 as a template with a primer pair pIBGvleu2-CBA-F (SEQ. ID. No. 30) and pIBGvncr-CBA-R (SEQ. ID. No. 31). The obtained plasmid was named pINCCB-Sed1.
A DNA fragment containing the promoter region of Saccharomyces cerevisiae-derived cell surface-localized protein gene Cwp2 (hereinafter, alternatively referred to as “CWP2” for the sake of convenience) was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair CWP2pv-CBA-F (SEQ. ID. No. 32) and CWP2ps-CBA-R (SEQ. ID. No. 33). This fragment was ligated, by one-step isothermal assembly, to a fragment amplified using a plasmid pISpBG-PG-Sed1 as a template with a primer pair pIBGsc2p-CBA-F (SEQ. ID. No. 34) and pIBGvc2p-CBA-R (SEQ. ID. No. 35). The obtained plasmid was named pIC2BG-PG-Sed1.
A DNA fragment containing the coding region of Saccharomyces cerevisiae-derived cell surface-localized protein gene CWP2 was prepared through amplification by PCR using a genome of Saccharomyces cerevisiae BY4741 strain as a template with a primer pair CWP2α-PG-F (SEQ. ID. No. 36) and CWP2α-BsrGI-R (SEQ. ID. No. 37).
A DNA fragment (BGL1-CWP2) in which the DNA fragment containing the coding region of the CWP2 gene was ligated in-frame downstream of the BGL1 gene fragment was prepared through amplification by PCR using a mixture of the BGL1 gene fragment and the DNA fragment containing the coding region of CWP2 gene as a template with a primer pair BGL1-NcoI-F (SEQ. ID. No. 13) and CWP2α-BsrGI-R (SEQ. ID. No. 37).
The BGL1-CWP2 fragment was treated with NcoI and BsrGI, and was ligated in-frame downstream of the secretion signal in plasmids pISpBG-PG-Sed1 and pIC2BG-PG-Sed1 treated in a similar manner. The obtained plasmids were respectively named pISpBG-PG-Cwp2 and pIC2BG-PG-Cwp2.
Example 2: Preparation of Cellulase-Displaying Yeast
A plasmid (pIBG-PG-Agα1 or pIEG-Agα1) for any of BGL1 and EGII genes was treated with NdeI, and was each provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. These recombinant strains are referred to as Gap-Agα1 recombinant strains for respective genes.
A plasmid (pIBG-PG-Sed1 or pIEG-Sed1) for any of BGL1 and EGII genes was treated with NdeI, and was each provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. These recombinant strains are referred to as Gap-Sed1 recombinant strains for respective genes.
A plasmid (pISpBG-PG-Agα1 or pISpEG-Agα1) for any of BGL1 and EGII genes was treated with NdeI, and was each provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. These recombinant strains are referred to as Sed1-Agα1 recombinant strains for respective genes.
A plasmid (pISpBG-PG-Sed1 or pISpEG-Sed1) for any of BGL1 and EGII genes was treated with NdeI, and was each provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. These recombinant strains are referred to as Sed1-Sed1 recombinant strains for respective genes.
A plasmid (pISpBG-PG-Cwp2 or pIC2BG-PG-Cwp2) for BGL1 gene was treated with NdeI, and was each provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. These recombinant strains are referred to as Sed1-Cwp2 and Cwp2-Cwp2 recombinant strains for BGL1 gene, respectively.
A plasmid (pINCCB-Agα1) for CBHII gene was treated with NdeI, and was provided with the Gap-Agα1 recombinant strain (MATα leu2 met15 ura3 strain) for EGII gene to transform the strain by lithium acetate treatment procedure. This recombinant strain is referred to as an EGII-CBHII gene co-expression type Gap-Agα1 recombinant strain.
A plasmid (pINCCB-Sed1) for CBHII gene was treated with NdeI, and was provided with the Sed1-Sed1 recombinant strain (MATα leu2 met15 ura3 strain) for EGII gene to transform the strain by lithium acetate treatment procedure. This recombinant strain is referred to as an EGII-CBHII gene co-expression type Sed1-Sed1 recombinant strain.
A vector plasmid pRS403 (HIS3 yeast expression vector: Agilent Technologies), free from any polynucleotide for cell surface expression, was treated with NdeI, and was provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. This recombinant strain is referred to as an empty vector-introduced strain.
Example 3: Examination for β-Glucosidase Activity, 1
The Gap-Agα1 recombinant strain, Gap-Sed1 recombinant strain, Sed1-Agα1 recombinant strain, and Sed1-Sed1 recombinant strain for BGL1 gene were examined for β-glucosidase (BGL) activity.
Cells were inoculated to 5 mL of SD medium (supplemented with leucine, methionine, and uracil) and cultured at 30° C. and 180 rpm for 18 hours (pre-culture), and then were inoculated to 50 mL of 1×YPD medium (initial OD600=0.05) and cultured at 30° C. and 150 rpm (main culture). The culture solutions were collected every 24 hours after the start of the main culture, and β-glucosidase activities of the cells were measured as follows:
(1) wash the cells twice with distilled water;
(2) prepare 500 μL of reaction mixture (composition: 100 μL of 10 mM pNPG (p-nitrophenyl-β-D-glucopyranoside) (final concentration 2 mM); 50 μL of 500 mM sodium citrate buffer solution (pH 5.0) (final concentration 50 mM); 250 μL of distilled water; and 100 μL of yeast cell suspension) (final cell concentration 1 to 10 g wet cells/L)), and allow it to be at 500 rpm and 30° C. for 10 minutes for the reaction;
(3) after the end of the reaction, stop the reaction by adding 500 μL of 3M Na2CO3; and
(4) centrifuge the mixture at 10,000 g for 5 minutes, and then measure the absorbance at 400 nm, ABS400, of the supernatant. One unit of enzyme activity was defined as the amount of enzymes required to liberate 1 μmol of pNP (p-nitrophenol) per minute.
The result is shown in FIG. 1. Symbols in FIG. 1 are as follows: white circles, Gap-Agα1 recombinant strain (Gap-Agα1 type); white diamonds, Gap-Sed1 recombinant strain (Gap-Sed1 type); white triangles, Sed1-Agα1 recombinant strain (Sed1-Agα1 type); and white squares, Sed1-Sed1 recombinant strain (Sed1-Sed1 type). As shown in FIG. 1, it was seen that the Sed1-Sed1 recombinant strain exhibited a BGL activity remarkably higher than that of any of the Gap-Agα1 recombinant strain, the Gap-Sed1 recombinant strain, and the Sed1-Agα1 recombinant strain. In this manner, it is seen that when the promoter and the anchor of Sed1 (SED1) are used in combination, synergistic effects are obtained, and BGL activities are significantly improved compared with the case of using only one of them.
Example 4: Examination for Endoglucanase Activity
The Gap-Agα1 recombinant strain, Gap-Sed1 recombinant strain, Sed1-Agα1 recombinant strain, and Sed1-Sed1 recombinant strain for EGII gene were examined for endoglucanase (EG) activity.
Cells were inoculated to 5 mL of SD medium (supplemented with leucine, methionine, and uracil) and cultured at 30° C. and 180 rpm for 18 hours (pre-culture), and then were inoculated to 50 mL of 1×YPD medium (initial OD600=0.05) and cultured at 30° C. and 150 rpm (main culture). The culture solutions were collected 48 hours after the start of the main culture, and endoglucanase activities of the cells were measured as follows:
(1) wash the cells twice with distilled water;
(2) prepare 2500 μL of reaction mixture (composition: one tablet of Cellazyme C (manufactured by Megazyme); 250 μL of 500 mM sodium citrate buffer solution (pH 5.0) (final concentration 50 mM); 2000 μL of distilled water; and 250 μL of yeast cell suspension (final cell concentration 10 g wet cells/L)), and allow it to stand at 38° C. for 4 hours for the reaction;
(3) after the end of the reaction, centrifuge the mixture at 10,000 g for 5 minutes, and then measure the absorbance at 590 nm, ABS590, of the supernatant.
The result is shown in FIG. 2. In FIG. 2, the horizontal axis indicates the results of the Gap-Agα1 recombinant strain (Gap-Agα1), the Gap-Sed1 recombinant strain (Gap-Sed1), the Sed1-Agα1 recombinant strain (Sed1-Agα1), and the Sed1-Sed1 recombinant strain (Sed1-Sed1) sequentially from the left, and the vertical axis indicates the light absorbance at 590 nm. As shown in FIG. 2, it was seen that the Sed1-Sed1 recombinant strain exhibited an EG activity remarkably higher than that of any of the Gap-Agα1 recombinant strain, the Gap-Sed1 recombinant strain, and the Sed1-Agα1 recombinant strain. In this manner, it is seen that, also in the case of EG, when the promoter and the anchor of Sed1 (SED1) are used in combination, synergistic effects are obtained, and activities are significantly improved compared with the case of using only one of them.
Example 5: Examination for β-Glucosidase Activity, 2
The Sed1-Sed1 recombinant strain, Gap-Agα1 recombinant strain, and Cwp2-Cwp2 recombinant strain for BGL1 gene were examined for β-glucosidase (BGL) activity, as in Example 3.
The result is shown in FIG. 3. Symbols in FIG. 3 are as follows: white circles, Gap-Agα1 recombinant strain (Gap-Agα1 type); white squares, Sed1-Sed1 recombinant strain (Sed1-Sed1 type); and white triangles, Cwp2-Cwp2 recombinant strain (Cwp2-Cwp2 type). As shown in FIG. 3, it was seen that the Sed1-Sed1 recombinant strain exhibited a BGL activity remarkably higher than that of the Gap-Agα1 recombinant strain, and, furthermore, the Cwp2-Cwp2 recombinant strain also exhibited a remarkably increased BGL activity. In this manner, when an anchor and a promoter were derived from the same gene, the activity improving effect obtained by use in combination was not limited to the case of using a combination of those derived from Sed1, and was also seen in the case of using a combination of those derived from other cell surface-localized proteins such as Cwp2 (CWP2).
Example 6: Examination for Hydrolysis of Hydrothermally Processed Rice Straw
The EGII-CBHII gene co-expression type Sed1-Sed1 recombinant strain, EGII-CBHII gene co-expression type Gap-Agα1 recombinant strain, and an empty vector-introduced strain were examined for effect on hydrolysis of hydrothermally processed rice straw.
Cells were inoculated to 10 mL of SD medium (supplemented with methionine and uracil) and cultured at 30° C. and 180 rpm for 18 hours (pre-culture), and then were inoculated to 500 mL of 1×YPD medium (initial OD600=0.05) and subjected to stationary culture at 30° C. (main culture). The culture solutions were collected 48 hours after the start of the main culture, and the cells were washed twice with distilled water and used for hydrolysis treatment.
Rice straw was treated with water at 130 to 300° C. and about 10 MPa, and a solid content was collected therefrom and used as hydrothermally processed rice straw.
The hydrolysis treatment was performed under the following conditions:
















Hydrothermally processed rice straw
100 g dry weight/L



Yeast extract
10 g/L



Peptone
20 g/L



Sodium citrate buffer solution
50 mM (pH 5.0)



Yeast cells
100 g wet cell weight/L



Total
10 mL










These were placed in a rotary fermenter, and were incubated at 38° C. and 35 rpm for 96 hours, without adding a commercially available exogenous enzyme.
With the empty vector-introduced strain (no cell surface display), no change was seen in the hydrothermally processed rice straw even after 96 hours. With the EGII-CBHII gene co-expression type Gap-Aged recombinant strain, an increase in the fluidity due to hydrolysis was gradually seen after 48 hours and thereafter. With the EGII-CBHII gene co-expression type Sed1-Sed1 recombinant strain, a significant increase in the fluidity was seen in 15 hours. With such a Sed1-Sed1 recombinant strain, hydrothermally processed rice straw was hydrolyzed to significantly increase the fluidity.
Example 7: Examination for β-Glucosidase Activity, 3
The Sed1-Sed1 recombinant strain, Cwp2-Cwp2 recombinant strain, and Sed1-Cwp2 recombinant strain for BGL1 gene were examined for β-glucosidase (BGL) activity, as in Example 3.
The result is shown in FIG. 4. Symbols in FIG. 4 are as follows: white squares, Sed1-Sed1 recombinant strain (Sed1-Sed1 type); white triangles, Cwp2-Cwp2 recombinant strain (Cwp2-Cwp2 type); and black triangles, Sed1-Cwp2 recombinant strain (Sed1-Cwp2 type). As shown in FIG. 4, it was seen that the Sed1-Cwp2 recombinant strain did not exhibit an increase in the activity as much as that of the Sed1-Sed1 recombinant strain and the Cwp2-Cwp2 recombinant strain, but exhibited an activity increased compared with that of the Gap-Agα1 recombinant strain.
Example 8: Examination for Cell-Surface β-Glucosidase Activity in SED1 Gene Disrupted Strain
In this example, the β-glucosidase-displaying yeasts prepared from BY4741 strain and SED1 gene disrupted strain thereof (BY4741 SED1Δ strain) using the coding region and promoter of the gene Sed1 were examined for β-glucosidase (BGL) activity.
The BY4741 strain and the BY4741 SED1Δ strain were transformed with a plasmid pISpBG-PG-Sed1 for BGL1 gene, as in Example 2. The BY4741 SED1Δ strain was obtained from Yeast Knockout Collection Parental Strain-BY4741 (Open Biosystems). The obtained recombinant strains, i.e., the Sed1-Sed1 recombinant BY4741 SED1Δ strain and the Sed1-Sed1 recombinant BY4741 strain were examined for β-glucosidase (BGL) activity, as in Example 3.
The result is shown in FIG. 5. Symbols in FIG. 5 are as follows: black circles, Sed1-Sed1 recombinant BY4741 SED1Δ strain (SED1Δ strain); and black squares, Sed1-Sed1 recombinant BY4741 strain (BY4741 strain). As shown in FIG. 5, the Sed1-Sed1 recombinant BY4741 SED1Δ strain exhibited a maximum cell-surface BGL activity increased compared with that of the Sed1-Sed1 recombinant BY4741 strain, and exhibited a remarkably increased BGL activity especially after culture for 72 hours and thereafter.
Example 9: Examination for Cell-Surface β-Glucosidase Activity in SSD1 Gene Disrupted Strain
In this example, the β-glucosidase-displaying yeasts prepared from BY4741 strain and SSD1 gene disrupted strain thereof (BY4741 SSD1Δ strain) using the coding region and promoter of the gene Sed1 were examined for β-glucosidase (BGL) activity.
The BY4741 strain and the BY4741 SSD1Δ strain were transformed with a plasmid pISpBG-PG-Sed1 for BGL1 gene, as in Example 2. The BY4741 SSD1Δ strain was obtained from Yeast Knockout Collection Parental Strain-BY4741 (Open Biosystems). The obtained recombinant strains, i.e., the Sed1-Sed1 recombinant BY4741 SSD1Δ strain and the Sed1-Sed1 recombinant BY4741 strain were examined for β-glucosidase (BGL) activity, as in Example 3.
The result is shown in FIG. 6. Symbols in FIG. 6 are as follows: black triangles, Sed1-Sed1 recombinant BY4741 SSD1Δ strain (SSD1Δ strain); and black squares, Sed1-Sed1 recombinant BY4741 strain (BY4741 strain). As shown in FIG. 6, the Sed1-Sed1 recombinant BY4741 SSD1Δ strain did not exhibit a significant increase in the maximum cell-surface BGL activity compared with that of the Sed1-Sed1 recombinant BY4741 strain, but exhibited a BGL activity somewhat increased after culture for 48 hours. Furthermore, the total cell amount of the Sed1-Sed1 recombinant BY4741 SSD10 strain increased to 1.16 times after culture for 48 hours and to 1.26 times after 96 hours compared with the Sed1-Sed1 recombinant BY4741 strain, and thus, the total BGL activity exhibited by the cells obtained by the culture was significantly increased.
Example 10: Examination for Cell-Surface β-Glucosidase Activity in Double-Disrupted Strain
In this example, the β-glucosidase-displaying yeasts prepared from BY4741 strain having disrupted SED1 and SSD1 genes (double-disrupted strain), as well as BY4741 strain, BY4741 SED10 strain, and BY4741 SSD1Δ strain using the coding region and promoter of the gene Sed1 were examined for were examined for β-glucosidase (BGL) activity.
The double-disrupted strain of the Sed1-Sed1 recombinant BY4741 strain was prepared as follows. About 1.1 kb of DNA fragment having a Zeocin-resistant gene and 30b sequences homologous with regions upstream and downstream of the SED1 gene respectively arranged at both termini was prepared by PCR using a plasmid pTEF1/Zeo (obtained from Invitrogen) as a template with a primer pair SED1d-zeo-F1 (SEQ. ID. No. 40) and SED1d-zeo-R1 (SEQ. ID. No. 41). Furthermore, about 1.2 kb of DNA fragment having a Zeocin-resistant gene and 80b sequences homologous with regions upstream and downstream of the SED1 gene respectively arranged at both termini was prepared through amplification again by PCR using the above-described fragment as a template with a primer pair SED1d-zeo-F2 (SEQ. ID. No. 42) and SED1d-zeo-R2 (SEQ. ID. No. 43). This DNA fragment was provided with the Sed1-Sed1 recombinant SSD1Δ strain obtained in Example 9 to transform the strain by lithium acetate treatment procedure, after which a Zeocin-resistant strain was selected so that a SED1 and SSD1 double-disrupted strain was obtained. The obtained recombinant strain was referred to as a Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ strain.
The Sed1-Sed1 recombinant BY4741 strain, Sed1-Sed1 recombinant BY4741 SED1Δ strain, Sed1-Sed1 recombinant BY4741 SSD1Δ strain, and Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ strain were examined for β-glucosidase (BGL) activity, as in Example 3.
The result is shown in FIG. 7. Symbols in FIG. 7 are as follows: white diamonds, Sed1-Sed1 recombinant BY4741 strain (BY4741); black squares, Sed1-Sed1 recombinant BY4741 SED1Δ strain (BY4741 SED1Δ); black triangles, Sed1-Sed1 recombinant BY4741 SSD1Δ strain (BY4741 SSD1Δ); and black circles, Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ strain (BY4741 SED1ΔSSD1Δ). As shown in FIG. 7, it was found that a maximum value and an increase rate of the cell-surface BGL activity for the double-disrupted strain as a host (Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ strain) expressing the SED1 type cell surface display cassette were higher than those for the SED1 single disrupted strain as a host (Sed1-Sed1 recombinant BY4741 SED1Δ strain). Furthermore, the total cell amount of the Sed1-Sed1 recombinant BY4741 SED1ΔSSD1Δ strain increased to 1.10 times after 48 hours and to 1.20 times after 96 hours compared with the Sed1-Sed1 recombinant BY4741 strain, and the effects were seen substantially similar to those in the case where the SSD1 single disrupted strain was used as a host (Sed1-Sed1 recombinant BY4741 SED1Δ strain).
Example 11: Examination for Ethanol Productivity
The Sed1-Sed1 recombinant strain and Gap-Agα1 recombinant strain for EGII gene were examined for ethanol productivity.
In this example, two types of EGII-displaying strains expressing the EGII gene on the cell surface, that is, EGII-displaying Sed1-Sed1 recombinant strain and EGII-displaying Gap-Agα1 recombinant strain, and an empty vector-introduced strain (no cell surface display) were used. This examination was conducted as in the procedure in Example 6, except that a culture solution having the following composition was used for fermentation culture:













Hydrothermally processed rice straw
100 g dry weight/L


Yeast extract
10 g/L


Peptone
20 g/L


Sodium citrate buffer solution
50 mM (pH 5.0)


Yeast cells
100 g wet cell weight/L


Exogenous enzyme (Ctec2: manufactured
1 FPU/10 mL


by Novozymes)


Total
10 mL









The result is shown in FIG. 8. Symbols in FIG. 8 are as follows; black circles, EGII-displaying Sed1-Sed1 recombinant strain (Sed1-Sed1 type); black triangles, EGII-displaying Gap-Agα1 recombinant strain (Gap-Agα1 type); and black squares, empty vector-introduced strain (no cell surface display). As shown in FIG. 8, the EGII-displaying Gap-Agα1 recombinant strain was not so much different from the empty vector-introduced strain of no cell surface display in terms of the rate and yield of the ethanol production in simultaneous saccharification and fermentation using hydrothermally processed rice straw, whereas the Sed1-Sed1 recombinant strain remarkably increased in the rate and yield.
Example 12: Preparation of Vector for Amylase Cell Surface Display
The preparation of Rhizopus oryzae-derived glucoamylase gene, and Streptococcus bovis-derived α-amylase gene were carried out as set forth below.
A glucoamylase gene fragment was prepared through amplification by PCR using pδU-PGGluRAG (a vector for cell surface expression of glucoamylase having a 3′ half region of α-agglutinin gene: Non-Patent Document 9) as a template with a primer pair pIBGvsp-CBA-F (SEQ. ID. No. 11) and GA-CBA-R (SEQ. ID. No. 44).
A gene fragment containing a fragment encoding the secretion signal peptide of yeast Saccharomyces cerevisiae a factor and Streptococcus bovis-derived α-amylase gene was prepared through amplification by PCR using pUPGSBAAG (a vector for cell surface expression of α-amylase having a 3′ half region of α-agglutinin gene: Non-Patent Document 10) as a template with a primer pair AA-CBA-F (SEQ. ID. No. 45) and AA-CBA-R (SEQ. ID. No. 46) for α-amylase.
A gene fragment containing the glucoamylase gene, or a gene fragment containing the α factor secretion signal peptide coding fragment and the α-amylase gene was ligated, by one-step isothermal assembly, to a fragment amplified respectively using a vector plasmid pINCCB-Sed1 or pISpBG-PG-Sed1 as a template with a primer pair SED1aga-CBA-F (SEQ. ID. No. 47) and SED1p-CBA-R (SEQ. ID. No. 10), or SED1aaa-CBA-F (SEQ. ID. No. 48) and SED1p-CBA-R2 (SEQ. ID. No. 49). The obtained plasmids were named pINCGA-Sed1 and pISpAA-Sed1.
Example 13: Preparation of Amylase-Displaying Yeast
A plasmid (pISpAA-Sed1) for α-amylase gene was treated with NdeI, and was provided with yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) to transform the strain by lithium acetate treatment procedure. This recombinant strain is referred to as a Sed1-Sed1 recombinant strain for α-amylase gene.
A plasmid (pINCGA-Sed1) for glucoamylase gene was treated with NdeI, and was provided with the Sed1-Sed1 recombinant strain (MATα leu2 met15 ura3 strain) for α-amylase gene to transform the strain by lithium acetate treatment procedure. This recombinant strain is referred to as an α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain.
Example 14: Examination for α-Amylase and Glucoamylase Activities
The α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain was examined for α-amylase and glucoamylase activities.
Cells were inoculated to 5 mL of SD medium (supplemented with methionine and uracil) and cultured at 30° C. and 180 rpm for 18 hours (pre-culture), and then were inoculated to 50 mL of 1×YPD medium (initial OD600=0.05) and cultured at 30° C. and 150 rpm (main culture). The culture solution was collected every 24 hours after the start of the main culture, and the cells were washed twice with distilled water. The α-amylase and glucoamylase activities of the cells were measured respectively using an α-amylase assay kit (manufactured by Kikkoman Corporation) and a saccharification assay kit (manufactured by Kikkoman Corporation), at pH 5.0 and 37° C.
The result is shown in FIG. 9. In FIG. 9, white diamonds indicate the measurement result of α-amylase activity, and white squares indicate the measurement result of glucoamylase activity. As shown in FIG. 9, it was seen that the α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain exhibit a high α-amylase activity and a high glucoamylase activity together. In this manner, starch-degrading enzyme (α-amylase and glucoamylase) were demonstrated to be displayed on the cell surface, same as cellulose-degrading enzymes (endoglucanase, cellobiohydrolase, and β-glucosidase).
Example 15: Examination for Simultaneous Saccharification and Fermentation of Starch
The α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain was examined for the productivity of ethanol from raw starch.
Cells were inoculated to 10 mL of SD medium (supplemented with methionine and uracil) and cultured at 30° C. and 180 rpm for 18 hours (pre-culture), and then were inoculated to 500 mL of 1×YPD medium (initial OD600=0.05) and cultured at 30° C. and 150 rpm (main culture). The culture solution was collected 72 hours after the start of the main culture, and the cells were washed twice with distilled water and used for simultaneous saccharification and fermentation.
Corn-derived starch (manufactured by Wako Pure Chemical Industries, Ltd.) was used as raw starch.
The hydrolysis treatment was performed under the following conditions:
















Raw starch
200 g dry weight/L



Yeast extract
10 g/L



Peptone
20 g/L



Yeast cells
50 g wet cell weight/L



Total
5 mL










These were placed in a rotary fermenter, and were incubated at 30° C. and 35 rpm for 120 hours, without adding a commercially available exogenous enzyme.
The result is shown in FIG. 10. With the α-amylase-glucoamylase gene co-expression type Sed1-Sed1 recombinant strain, raw starch was hydrolyzed and directly converted to ethanol by the yeast cells alone without adding a commercially available exogenous enzyme.
INDUSTRIAL APPLICABILITY
Provided is a polynucleotide for cell surface expression that allows for the production of a yeast displaying a protein such as an enzyme on the cell surface with a high activity. A yeast into which this polynucleotide has been introduced can express a protein on the cell surface with a high activity. For example, if the protein is a cellulose-degrading enzyme, it is applicable to produce biofuels (ethanol, butanol, isoprenoid, etc.) and chemical polymer materials (isopropanol, amino acid, organic acid, quinones, etc.) from cellulosic biomass. Effective use of biomass not only reduces the environmental load but also suppresses CO2 emissions due to use of fossil resources.

## Claims

1. A polynucleotide for cell surface expression, comprising various heterologous elements: a) an SED1 promoter of SEQ ID NO: 5, b) a sequence encoding a secretion signal peptide of Rhizopus oryzae-derived glucoamylase or Saccharomyces cerevisiae α factor, c) a sequence encoding an intended protein, and d) a sequence encoding a cell surface-localized protein SED1 of SEQ ID NO: 2 or a cell membrane-binding domain thereof including positions 110 to 338 of SEQ ID NO: 2, said intended protein being a protein selected from the group consisting of enzyme, antibody, ligand and fluorescent protein.

2. An expression vector, comprising the polynucleotide for cell surface expression of claim 1.

3. A recombinant yeast into which the polynucleotide for cell surface expression of claim 1 has been introduced.

4. The recombinant yeast of claim 3, obtained from a host yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient.

5. The recombinant yeast of claim 4, obtained from a host yeast in which SED1 and SSD1 are deficient.

6. The recombinant yeast of claim 3, displaying at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface.

7. A method for producing ethanol, comprising:
performing fermentation culture of the recombinant yeast of claim 6.

8. A recombinant yeast into which the expression vector of claim 2 has been introduced.

9. The recombinant yeast of claim 8, obtained from a host yeast in which at least one selected from the group consisting of SED1 and SSD1 is deficient.

10. The recombinant yeast of claim 9, obtained from a host yeast in which SED1 and SSD1 are deficient.

11. The recombinant yeast of claim 8, displaying at least one enzyme selected from the group consisting of cellulose-degrading enzymes and starch-degrading enzymes on the cell surface.