JP7319652B2 - protein cell surface expressing yeast - Google Patents

Description

The present invention relates to protein cell surface expressing yeast.

For example, for the production of ethanol from lignocellulosic biomass, surface display techniques have been used to express proteins such as cellulolytic enzymes in yeast and efficiently display them on the surface of the cells (e.g., Patent Documents 1 and 2). In order to improve such surface display technology, it has been conventional to increase the amount of protein expression using a high-expression promoter, or to efficiently transport heterologous proteins to the cell surface using a signal sequence with high secretion ability. Approaches have been taken to improve expression cassettes containing proteins for display purposes.

In the protein surface-expressing yeast prepared by such an approach, the activity of the protein on the cell surface can be improved to a predetermined level. However, from the point of view of practicality, for example, for the purpose of improving the industrial productivity of the target product such as ethanol, it is difficult to say that the needs have been sufficiently met, and further improvement of the protein activity of the surface layer is required. there is

WO2014/157141 WO2016/017736

An object of the present invention is to provide a protein cell surface-expressing yeast capable of further improving the activity of the protein on the cell surface without adhering only to the expression cassette improvement approach of the conventional cell surface display technology.

The present invention provides a transformed yeast that expresses a protein on the cell surface,
Function of at least one cell wall-related gene in a host yeast carrying at least one cell wall-related gene selected from the group consisting of CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene, CWP1 gene, and corresponding genes deleted, and has introduced a polynucleotide comprising a promoter, a secretory signal sequence, DNA encoding the protein, DNA encoding the anchor domain, and a terminator;
A transformed yeast is provided.

In one embodiment, the transformed yeast lacks the function of at least one of the CCW12 gene and the CCW14 gene.

In one embodiment, the transformed yeast lacks the functions of all of the cell wall-associated genes of CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene and CWP1 gene.

In one embodiment, the transformed yeast further lacks the function of the SED1 gene.

In one embodiment, the protein is an enzyme.

In one embodiment, the enzyme is a cellulolytic enzyme.

In one embodiment, the host yeast is Saccharomyces yeast.

The present invention provides a method for producing a transformed yeast that expresses a protein on the cell surface, the method comprising:
At least one cell wall of the host yeast in the host yeast carrying at least one cell wall-associated gene selected from the group consisting of CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene, CWP1 gene, and corresponding genes. and introducing into said host yeast a polynucleotide comprising a promoter, a secretory signal sequence, DNA encoding said protein, DNA encoding an anchor domain and a terminator.

The present invention provides an enzymatic preparation containing the transformed yeast.

In one embodiment, the enzymatic agent is used for ethanol production using biomass.

The present invention provides a method for producing ethanol, which method comprises the step of culturing the above transformed yeast in a medium containing β-1,4-linked glucose polysaccharides.

INDUSTRIAL APPLICABILITY According to the present invention, by changing the cell wall structure of the cell surface layer of yeast, the activity of proteins in the cell surface layer can be further improved. Furthermore, according to the present invention, it is possible to provide, for example, yeast in which a cellulolytic enzyme useful for ethanol production from lignocellulosic biomass is expressed on the cell surface.

Surface display transformed yeast obtained by introducing a plasmid containing expression cassette X8 into Saccharomyces cerevisiae BY4741 strain and its 5-gene knockout strain as hosts (A: BY-EG-SSSD strain and B: GPIm-EGSD strain) 2 is an electron micrograph showing the morphology of the cell wall of each. (A) of surface display transformed yeast (BY-BG-SSSD strain and GPIm-BGSD strain) obtained by introducing a plasmid containing expression cassette X7 using Saccharomyces cerevisiae strain BY4741 and its 5-gene knockout strain as hosts. 1 is a graph showing changes over time in β-glucosidase activity of cells and (B) growth of cells. Cell end of surface display transformed yeast (BY-EG-SSSD strain and GPIm-EGSD strain) obtained by introducing a plasmid containing expression cassette X8 into Saccharomyces cerevisiae strain BY4741 and its 5-gene knockout strain as hosts Fig. 3 is a graph showing glucanase activity; Saccharomyces cerevisiae strain BY4741, a 5-gene knockout strain, and a strain in which only 2 (CCW12 and CCW14) or 3 (TIP1, CWP1, and DAN1) of the 5 genes are knocked out were used as hosts to generate a plasmid containing the expression cassette X7. (A) Cell β-glucosidase of surface display transformed yeast (BY-BG-SSSD strain, GPIm-BGSD strain, ccw12/ccw14-BGSD strain, tip1/cwp1/dan1-BGSD strain) obtained by introduction It is a graph which shows a time-dependent change of activity and (B) bacterial growth. Surface display transformed yeast (GPIm-BGSD strain) obtained by introducing a plasmid containing expression cassette X7 into a 5-gene knockout strain as a host, and a transformant strain (GPIm-sed1- BGSD strain) (A) β-glucosidase activity of cells and (B) time course of cell growth. Saccharomyces cerevisiae BY4741 strain, CCW12 and CCW14 2-gene knockout strains, and 5-gene knockout strains were used as hosts, and surface-displaying transformed yeast obtained by introducing a plasmid containing the expression cassette X7 (BY-BG-SSSD strain, ccw12 2 is a graph showing changes over time in the concentration of (A) cellobiose and (B) ethanol in the culture solution during cultivation of the /ccw14-BGSD strain and the GPIm-BGSD strain) in a cellobiose-containing medium.

The present invention will now be described in detail.

(1. Cell Surface Presentation Method)
First, the cell surface display method adopted for the protein cell surface-expressing yeast of the present invention will be described. This cell surface display method is carried out using a polynucleotide containing a promoter, a secretory signal sequence, a DNA encoding a protein of interest to be displayed on the cell surface (“protein of interest”), a DNA encoding an anchor domain, and a terminator.

(1.1 DNA Encoding Anchor Domain)
The term “anchor domain” refers to a domain having an anchoring activity that immobilizes a target protein on the cell surface of yeast. As the "anchor domain", for example, a cell surface localized protein can be used. A “cell surface-localized protein” refers to a protein that is immobilized or attached or adhered to the cell surface and localized there. A lipid-modified protein is known as a cell surface-localized protein, and is fixed to the cell membrane by covalently binding the lipid to a membrane component.

Representative examples of cell surface localized proteins include GPI (glycosyl phosphatidyl inositol): ethanolamine phosphate-6 mannose α-1,2 mannose α-1,6 mannose α-1,4 glucosamine α-1, Glycolipid anchor proteins having a basic structure of 6-inositol phospholipids can be mentioned. A GPI-anchored protein has a glycolipid, GPI, at its C-terminus, and this GPI binds to the cell membrane surface by covalently bonding with PI (phosphatidyl inositol) in the cell membrane.

Binding of GPI to the C-terminus of a GPI-anchored protein is performed, for example, as follows. After transcription and translation, GPI-anchored proteins are secreted into the lumen of the endoplasmic reticulum by the action of a secretory signal present on the N-terminal side. A region called a GPI-anchor attachment signal that is recognized when the GPI-anchor binds to the GPI-anchor protein exists in the C-terminus of the GPI-anchor protein or in the region near it. In the lumen of the endoplasmic reticulum and the Golgi apparatus, this GPI-anchor attachment signal region is cleaved and GPI binds to the newly generated C-terminus.

GPI-linked proteins are transported to the cell membrane by secretory vesicles and anchored to the cell membrane by covalent binding of GPI to PI in the cell membrane. Furthermore, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), incorporated into the cell wall, and presented on the cell surface in a cell wall-anchored state.

Methods of cell surface display include, for example, methods using GPI anchoring regions. In this method, the "GPI anchoring region" can be a DNA encoding the entire GPI anchor protein, which is a cell surface localized protein, or a region containing the GPI anchor attachment signal region, which is its cell wall binding region. In this method, the entire GPI-anchored protein or a region containing the GPI-anchor attachment signal region, which is the cell wall binding region thereof, is used as the “anchor domain”. Methods using GPI-anchored regions utilize GPI-anchor attachment signals for cell surface presentation. The cell wall binding domain (GPI anchor attachment signal domain) is usually the C-terminal domain of the cell surface localized protein. The cell wall-binding region may contain the GPI-anchor attachment signal region, and may contain any other portion of the GPI-anchor protein as long as it does not inhibit the enzymatic activity of the fusion protein.

The GPI-anchored protein may be any protein that functions in yeast cells. Such GPI-anchored proteins include, for example, SED1, α- or a-agglutinin (AGα1, AGA1), TIP1, FLO (eg FLO1), CWP1 and CWP2. As the anchor domain, SED1 or its cell wall binding domain (GPI anchor attachment signal domain) is preferably used. A region containing 320 amino acids from the C-terminus of α-agglutinin is also preferably used as the anchor domain, and the DNA encoding this region is also referred to as the 3′-terminal half region of the α-agglutinin gene.

The SED1 gene (Sed1) can be obtained using a method commonly used by those skilled in the art, for example, based on the sequence information registered in GenBank (GenBank accession number NM — 001180385; NCBI Gene ID: 851649). The nucleotide sequence of the protein coding region of Sed1 is shown in SEQ ID NO: 11 (the sequence without the initiation methionine is shown in SEQ ID NO: 47), and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 12 (the sequence without the initiation methionine is (shown in SEQ ID NO: 48). The cell wall binding region of SED1 is, for example, the region containing positions 110 to 338 of SEQ ID NO:48. The DNA encoding SED1 may be a polynucleotide encoding the full length of the amino acid sequence of SEQ ID NO: 48, or a partial sequence (for example, positions 110 to 338 of SEQ ID NO: 48) as long as it does not impair the anchor function. sequence (including amino acid sequence)).

Here, the protein or peptide described herein consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the disclosed amino acid sequence, and has the desired function or effect in the present invention. It may be a protein or peptide substantially comprising, and DNA or polynucleotides may include those encoding such proteins or peptides. Any one type of amino acid mutation (eg, deletion, substitution, or addition) to the disclosed amino acid sequence may be used, or two or more types may be combined. Moreover, the total number of these mutations is one or several, but is not particularly limited as long as it substantially has the desired function or effect, and may also depend on the size of the protein or peptide. The total number of mutations includes, for example, 1 or more and 10 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, or 1 or more and 2 or less. Not limited. Also, the total number of mutations can be within a range that satisfies sequence identity, eg, as described below. Examples of amino acid substitutions may be any substitutions as long as each function or effect is substantially retained. For example, conservative substitutions are included. Conservative substitutions specifically include substitutions within the following groups (i.e., between the amino acids shown in parentheses): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid) ), (asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine).

In other embodiments, the proteins or peptides described herein have amino acid sequences that have, for example, 70% or greater sequence identity to the disclosed amino acid sequences, and the desired in the present invention. and the polynucleotide may encode such a protein or peptide. Sequence identity in the amino acid sequence also refers to a protein that has an amino acid sequence that has, for example, 70% or more sequence identity with the disclosed amino acid sequence, and that substantially has the desired function or effect in the present invention. Or it may encode a peptide. The sequence identity in the amino acid sequence is also greater than 74%, greater than 78%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, 96% % or greater, 97% or greater, 98% or greater, or 99% or greater.

Sequence identity or similarity, as used herein, is a relationship between two or more proteins or two or more polynucleotides determined by comparing the sequences, as is known in the art. . Sequence "identity" refers to the identity between protein or polynucleotide sequences, as determined by an alignment between the protein or polynucleotide sequences, or optionally by an alignment between a series of partial sequences. Denotes the degree of sequence invariance. Also, "similarity" refers to the relationship between protein or polynucleotide sequences, as determined by an alignment between protein or polynucleotide sequences, or optionally by an alignment between a series of partial sequences. means the degree of correlation. More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as similarity in the sequence homology search results of BLAST, which will be described later. Methods to determine identity and similarity are preferably those designed to give the longest alignment between the sequences being compared. Methods to determine identity and similarity are provided as publicly available programs. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. ) can be used to determine Conditions for using software such as BLAST are not particularly limited, but default values are preferably used.

In still other embodiments, the proteins or peptides described herein may be encoded by DNA hybridized with DNA comprising a nucleotide sequence complementary to the DNA comprising the disclosed nucleotide sequence. Well, and the polynucleotide may comprise such hybridized DNA. Hybridization between a DNA consisting of a disclosed base sequence and a DNA consisting of a complementary base sequence is preferably carried out under stringent conditions.

Stringent conditions are, for example, conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, nucleic acids with high nucleotide sequence identity, i.e., disclosed nucleotide sequences, e.g. 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a complementary strand of DNA consisting of a base sequence having 99% or more identity hybridizes and a complementary strand of nucleic acid with lower homology does not hybridize. More specifically, the sodium salt concentration is, for example, 15 mM to 750 mM, 50 mM to 750 mM, or 300 mM to 750 mM, the temperature is, for example, 25° C. to 70° C., 50° C. to 70° C., or 55° C. to 65° C., and formamide It refers to conditions where the concentration is, for example, 0% to 50%, 20% to 50%, or 35% to 45%. Further, stringent conditions include washing conditions for the post-hybridization filter with a sodium salt concentration of, for example, 15 mM to 600 mM, 50 mM to 600 mM, or 300 mM to 600 mM, and a temperature of, for example, 50°C to 70°C, 55°C to 70°C, or 60°C to 65°C. DNA that hybridizes under stringent conditions means, for example, DNA-immobilized filters are used to perform hybridization at 65° C. in the presence of 0.7 to 1.0 M NaCl, followed by Examples include DNA that can be obtained by washing a filter at 65° C. in a 0.1- to 2-fold SSC solution (the composition of a 1-fold SSC solution is 150 mM NaCl and 15 mM sodium citrate). Hybridization can be performed by well-known methods such as those described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory (2001). Higher temperatures or lower salt concentrations increase stringency and allow the isolation of polynucleotides with higher homology (sequence identity).

In still other embodiments, the DNA or polynucleotide comprises a disclosed base sequence, e.g. % or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the identity, and the desired Polynucleotides that substantially have a function or effect are included.

A base sequence encoding a given amino acid sequence is obtained by substituting at least one base with another base without changing the amino acid sequence of the protein by substitution based on the degeneracy of the genetic code. You can also

As the "anchor domain", for example, when the cell surface-localized protein is a sugar chain-binding protein (eg, FLO (eg, FLO1)), the sugar chain-binding protein domain (International Publication No. 02/085935) is used. can also Yeast cell surface display methods using a sugar chain-binding protein domain include, for example, the N-terminal side, the C-terminal side, or both the N-terminal side and the C-terminal side of the sugar chain-binding protein domain of a cell surface localized protein. and a method of introducing into a host yeast a DNA designed to express a fusion protein in which a target protein is fused to. Proteins expressed from such introduced DNA and secreted to the outside of the cell membrane remain on the cell surface due to the interaction of multiple sugar chains possessed by the sugar chain-binding protein domain with sugar chains in the cell wall. obtain. Examples thereof include sugar chain binding sites of lectins and lectin-like proteins. Examples of sugar chain-binding protein domains typically include aggregation functional domains of GPI-anchored proteins. The term "aggregation functional domain of GPI-anchored protein" refers to a domain that is located on the N-terminal side of the GPI-anchored region, has multiple sugar chains, and is thought to be involved in aggregation between yeast cells. .

The DNA encoding the anchor domain is obtained by PCR using DNA extracted from microorganisms having these, nucleic acids derived from various cDNA libraries or genomic DNA libraries as templates, and primers designed based on known sequence information. Can be obtained as fragments. A DNA encoding an anchor domain may be obtained as a DNA fragment by hybridizing a nucleic acid derived from the library with a probe designed based on known sequence information. It can also be used by excising it as a DNA fragment from an existing vector containing DNA encoding the anchor domain. A DNA encoding an anchor domain may be synthesized as a DNA fragment by various nucleic acid sequence synthesis methods known in the art, such as chemical synthesis methods.

(1.2 Secretory signal sequence)
A "secretory signal sequence" is DNA that encodes a secretory signal peptide. Secretory signal peptides are peptides normally attached to the N-terminus of secretory proteins that are secreted extracellularly, including the periplasm. This peptide has a similar structure among organisms, for example, it consists of about 20 amino acids, has a basic amino acid sequence near the N-terminus, and contains many hydrophobic amino acids after that. Secretory signals are usually removed by being cleaved by signal peptidase when a secretory protein is secreted from inside the cell through the cell membrane to the outside of the cell.

In the present invention, any DNA can be used as long as it encodes a secretory signal peptide capable of extracellularly secreting a target protein from yeast, and the origin of the DNA is not limited. A DNA encoding a secretion signal peptide inherent in the target protein can also be used. It may be a nucleotide sequence containing mutations such as deletion, addition or substitution of one or more (for example, several) nucleotides in the nucleotide sequence encoding the native protein, as long as it exhibits the activity of a secretory signal, or It may be a nucleotide sequence encoding a protein consisting of an amino acid sequence containing mutations such as deletion, addition or substitution of one or more (eg, several) amino acids. With respect to the base sequence of the DNA encoding the secretory signal peptide and the amino acid sequence of the encoding protein, the sequence identity and hybridization conditions etc. described above apply.

For example, the secretory signal peptide of SED1 (described above), which is the major cell surface localized protein in the stationary phase of the yeast Saccharomyces cerevisiae. The amino acid sequence of this secretory signal peptide is shown in SEQ ID NO:45, and the nucleotide sequence of the DNA encoding it is shown in SEQ ID NO:44. Furthermore, for example, secretion signal peptides of glucoamylases such as Rhizopus oryzae, secretion signal peptides of glucoamylases of Aspergillus oryzae, secretion signal peptides of α- or a-agglutinin of the yeast Saccharomyces cerevisiae , a DNA encoding a secretory signal peptide of α-factor of the yeast Saccharomyces cerevisiae, and the like can be preferably used. The secretory signal peptide sequence of Rhizopus oryzae-derived glucoamylase is preferred in terms of secretory efficiency. DNA encoding the secretory signal peptide of Aspergillus oryzae-derived glucoamylase is also preferred.

The DNA encoding the secretory signal peptide is, for example, DNA extracted from a given yeast (eg, Saccharomyces cerevisiae) using primers designed based on known sequence information, various cDNA libraries, or genomic DNA libraries. A DNA fragment can be obtained by performing PCR using the derived nucleic acid as a template. A DNA encoding a secretory signal peptide can be obtained as a DNA fragment by hybridizing a nucleic acid derived from the library with a probe designed based on known sequence information. A DNA encoding a secretory signal peptide may be synthesized as a DNA fragment by various nucleic acid sequence synthesis methods known in the art, such as chemical synthesis methods. It can also be used by excising it as a DNA fragment (which may be in the form of an expression cassette) from an existing vector containing DNA encoding a secretory signal peptide.

For example, the secretory signal sequence of SED1 can be used with the coding region of the gene Sed1 of SED1 as the DNA encoding the anchor domain and the promoter of the gene Sed1 as the promoter.

(1.3 Target protein)
The type of target protein or its origin is not particularly limited. Types of target proteins include, for example, enzymes, antibodies, ligands, and fluorescent proteins. Examples of enzymes include cellulolytic enzymes, amylolytic enzymes, glycogenolytic enzymes, xylan degrading enzymes, chitinolytic enzymes, lipolytic enzymes, etc. More specifically, for example, endoglucanase, cellobiohydrolase, and β-glucosidase, amylase (eg, glucoamylase and α-amylase), lipase, and the like.

The DNA encoding the target protein is preferably a cDNA sequence excluding introns.

The DNA encoding the target protein may be a sequence encoding the entire length of the target protein, or may be a sequence encoding a partial region of the target protein as long as it exhibits the activity of the target protein. In addition, as described above, a nucleotide sequence containing mutations such as deletion, addition, or substitution of one or more (for example, several) nucleotides in the nucleotide sequence encoding the native protein as long as it exhibits the activity of the target protein. Alternatively, it may be a nucleotide sequence encoding a protein consisting of an amino acid sequence containing mutations such as deletion, addition or substitution of one or more (eg, several) amino acids. Regarding the base sequence of the DNA encoding the target protein and the amino acid sequence of the encoding protein, the sequence identity and hybridization conditions described above are applied.

The DNA (gene) encoding the target protein was designed based on known sequence information using DNA extracted from microorganisms that have or produce the protein, nucleic acids derived from various cDNA libraries or genomic DNA libraries as templates. It can be obtained as a nucleic acid fragment by PCR using primers. Such polynucleotides can be obtained as DNA fragments by hybridizing nucleic acids derived from the library with probes designed based on known sequence information. It can also be used by excising it as a DNA fragment (which may be in the form of an expression cassette) from an existing vector containing DNA encoding the target protein. Such genes can also be obtained by artificial synthesis, if necessary, based on sequences optimized in consideration of the codon frequency of the host.

As an example of the target protein, a cellulolytic enzyme will be described below.

Cellulolytic enzyme refers to any enzyme capable of cleaving β1,4-glycosidic bonds. The cellulolytic enzyme can be derived from any cellulolytic enzyme-producing fungus. Cellulose hydrolase-producing bacteria typically include the genus Aspergillus (e.g., Aspergillus aculeatus, Aspergillus niger, and Aspergillus oryzae), the genus Trichoderma (e.g., Trichoderma reesei), Clostridium (e.g. Clostridium thermocellum), Cellulomonas (e.g. Cellulomonas fimi and Cellulomonas uda), Examples thereof include microorganisms belonging to the genus Pseudomonas (for example, Pseudomonas fluorescence).

Although endoglucanase, cellobiohydrolase, and β-glucosidase will be described below as typical cellulolytic enzymes, cellulolytic enzymes are not limited to these.

Endoglucanases, also commonly referred to as cellulases, are enzymes that cleave cellulose from within the molecule to yield glucose, cellobiose, and cellooligosaccharides (“cellulose intramolecular cleavage”). There are five types of endoglucanases, termed endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase IV, and endoglucanase V, respectively. Although these are distinguished by the difference in amino acid sequence, they are common in that they have a cellulose intramolecular scission action. For example, Trichoderma reesei-derived endoglucanase (in particular, endoglucanase II: EGII) can be used, but is not limited thereto.

Cellobiohydrolases degrade to release cellobiose from either the reducing or non-reducing end of cellulose (“cellulose truncating”). There are two types of cellobiohydrolases, termed cellobiohydrolase I and cellobiohydrolase II, respectively. These distinctions are made by differences in amino acid sequences, but they are common in that they have a cellulose molecule terminal cleaving action. For example, Trichoderma reesei-derived cellobiohydrolase (especially cellobiohydrolase II: CBHII) can be used, but is not limited thereto.

β-Glucosidase is an exo-type hydrolase that cleaves glucose units from the non-reducing end of cellulose (“glucose unit cleavage”). β-Glucosidase can cleave the β1,4-glycosidic bond between the aglycone or sugar chain and β-D-glucose and hydrolyze cellobiose or cellooligosaccharide to produce glucose. β-Glucosidase is a representative example of an enzyme that can hydrolyze cellobiose or cellooligosaccharides. One type of β-glucosidase is currently known and designated as β-glucosidase-1. For example, β-glucosidase derived from Aspergillus aculatus (especially β-glucosidase 1: BGL1) can be used, but is not limited thereto.

Enzymes with different cellulose hydrolysis modes may be combined for good hydrolysis of cellulose. Enzymes that act in a variety of different cellulose hydrolysis modes, such as cellulose intramolecular scission, cellulose end scission, and glucose unit scission, can be appropriately combined. Examples of enzymes with respective hydrolysis modes include, but are not limited to, endoglucanases, cellobiohydrolases, and β-glucosidases. A combination of enzymes that hydrolyze cellulose differently can be selected, for example, from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases. Since it is desirable to be able to ultimately produce glucose, which is the constituent sugar of cellulose, it is preferable to include at least one enzyme capable of producing glucose. As enzymes capable of producing glucose, in addition to glucose unit-cleaving enzymes (eg, β-glucosidases), endoglucanases can also produce glucose. For example, in yeast, β-glucosidases, endoglucanases, and cellobiohydrolases can be secreted or surface-displayed.

(1.4 Promoter)
A promoter is a DNA having promoter activity, and the term "promoter activity" refers to the activity of binding a transcription factor to a promoter region to induce transcription. Promoters can be excised, for example, from bacterial cells, phages, etc. that possess a desired promoter region, using restriction enzymes. A DNA fragment of the promoter region can be obtained by amplifying the desired promoter region by PCR using primers provided with restriction enzyme recognition sites or overlapping sites with the cloning vector, if necessary. Alternatively, a desired promoter may be chemically synthesized based on already known nucleotide sequence information of the promoter region. As described above, the promoter may have a mutated base sequence such as deletion, addition or substitution of one or more (for example, several) nucleotides, as long as the desired function is achieved. good. The sequence identity, hybridization conditions, etc. described above are applied to the nucleotide sequence that constitutes this promoter.

The promoter can be any promoter as long as it has promoter activity in yeast. The promoter may be of the gene itself to be expressed, or may be derived from another gene. It may also be a promoter of a gene encoding a cell surface-localized protein used as an anchor domain. Examples include the SED1 promoter, TDH3 promoter, PGK1 promoter, CWP2 promoter, and TDH1 promoter.

The SED1 promoter is the promoter of the SED1 (described above) gene (Sed1), and its nucleotide sequence is shown in SEQ ID NO:46. The SED1 promoter is preferably used in combination with DNA encoding the coding region of the SED1 gene Sed1 as an anchor domain. Furthermore, it can be used in combination with the SED1 secretion signal sequence.

(1.5 Terminator)
A terminator is a DNA having terminator activity, and "terminator activity" refers to the activity of terminating transcription in the terminator region. Terminators can be excised, for example, from bacterial cells, phages, etc. that possess the desired terminator region, using restriction enzymes. A DNA fragment of the terminator region can be obtained by amplifying the desired terminator region by PCR using primers provided with a restriction enzyme recognition site or an insertion site for a cloning vector, if necessary. Alternatively, a desired terminator may be chemically synthesized based on already known base sequence information of the terminator region. A terminator may have a mutated base sequence such as deletion, addition or substitution of one or more (for example, several) nucleotides, as described above, as long as it fulfills the desired function. good. The sequence identity, hybridization conditions, and the like described above are applied to the nucleotide sequence that constitutes this terminator.

Terminators include, for example, α-agglutinin terminator, ADH1 (aldehyde dehydrogenase) terminator, TDH3 (glyceraldehyde-3'-phosphate dehydrogenase) terminator, DIT1 terminator and the like.

(1.6 Construction of Expression Cassette and Expression Vector)
In the present invention, the term "expression cassette" means that a DNA encoding a target protein and various regulatory elements for regulating its expression are combined in host microorganisms or cells so that the target protein can be expressed. DNA or polynucleotide that is operably linked to As used herein, "operably linked" means an expression cassette such that the DNA encoding the protein of interest is expressed under the control of a promoter, and optionally under the control of other regulatory elements. Alternatively, it means that each component contained in the expression vector is linked. Each component may be linked by further including a sequence such as a linker between those components as long as the target protein can be expressed.

In expression cassettes used in cell surface display technology (also referred to as "surface display cassettes"), a secretory signal sequence, a DNA encoding a target protein, and a DNA encoding an anchor domain function in host microorganisms or host cells. It can be connected in a state to obtain. A secretory signal sequence is located upstream of the DNA encoding the protein of interest. The arrangement of the DNA encoding the target protein and the DNA encoding the anchor domain is such that the expressed protein is fused to the N-terminal or C-terminal side of the anchor domain with the target protein, depending on the type of anchor domain. be done. The surface display cassette, for example, contains, from the 5′ end to the 3′ end, a secretory signal sequence, a DNA encoding a target protein, and a DNA encoding an anchor domain in the order described, or alternatively, the secretory signal sequence, the anchor domain, and the like. and a DNA encoding the target protein in the order listed. The surface display cassette further comprises a promoter upstream of the secretory signal sequence. In addition, the surface display cassette further includes a terminator downstream of the DNA encoding the target protein and the DNA encoding the anchor domain.

As used herein, the term "expression vector" refers to a vector into which a unit (expression cassette) for expressing DNA encoding a target protein has been inserted, and includes those into which DNA encoding the target protein has been inserted. Expression vectors may be plasmid vectors or artificial chromosomes. When yeast is used as a host, the form of plasmid is preferable because the vector can be easily prepared and yeast cells can be easily transformed. From the viewpoint of simplification of DNA acquisition, a shuttle vector between yeast and E. coli is preferred. Optionally, the vector may contain regulatory sequences (operators, enhancers, etc.). Such a vector has, for example, a yeast 2 μm plasmid replication origin (Ori) and a ColE1 replication origin, a yeast selectable marker (described below) and an E. coli selectable marker (drug resistance gene, etc.). ).

Any known marker can be used as the yeast selectable marker. For example, drug resistance genes, auxotrophic marker genes (e.g., genes encoding imidazole glycerol phosphate dehydrogenase (HIS3), genes encoding beta-isopropyl dehydrogenase malate (LEU2), O-acetylhomoserine O-acetylserine sulfide a gene encoding lylase (MET15), 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), and and the gene encoding alpha-aminoadipate reductase (LYS2). For example, an auxotrophic marker gene (eg, HIS3, LEU2, URA3, MET15 deficiency marker, etc.) can be preferably used.

Synthesis and ligation of DNA containing various sequences can be performed by methods commonly used by those skilled in the art. For binding of each component, for example, the DNA of each component provided with an appropriate restriction enzyme recognition sequence by PCR is cleaved with the restriction enzyme and ligated using ligase or the like, One-step isothermal assembly (Nature Methods, 2009, 6:343-345), or the In-Fusion method (Biotechniques, 2007, 43:354-359).

A plasmid containing a region (structural gene) encoding a target protein (e.g., enzymes such as endoglucanase, cellobiohydrolase, β-glucosidase, fluorescent proteins, or various antibodies) and expression regulatory sequences such as promoters and terminators, , can also be used by excising it in a form suitable for vector preparation and preparing an insert.

(2. Preparation of transformed yeast)
The transformed yeast of the present invention is produced by applying the cell surface display method described in 1 above to the host yeast. Since the transformed yeast of the present invention expresses a target protein on the cell surface and displays it on the cell surface, it can also be called "cell surface expressing yeast" or "cell surface displaying yeast".

The "host yeast" is not particularly limited as long as it belongs to Ascomycetous yeast. For example, Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwa Yeast such as Schwanniomyces, Komagataella and Yarrowia.

In the present invention, a "host yeast" is a host yeast carrying a given cell wall-related gene. The predetermined cell wall-associated genes are the CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene and CWP1 gene, their equivalent genes, and combinations thereof.

The method for producing a transformed yeast of the present invention comprises the step of deleting the function of at least one of the cell wall-related genes possessed by the host yeast in the host yeast carrying the predetermined cell wall-related gene; Including the step of introducing a polynucleotide as described into the host yeast. A polynucleotide may be in the form of an expression cassette or an expression vector. Either the step of deleting the function of the gene or the step of introducing the polynucleotide may be performed first. In particular, it is preferable to carry out the step of deleting the function of the gene first, because it becomes easy to switch the protein expressed on the cell surface depending on the application.

The CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene and CWP1 gene are known as yeast cell wall-related genes of the genus Saccharomyces. Sequence information for these genes is available from the Saccharomyces genome database (SGD). The CCW12 gene is also referred to as YLR110C and has an SGD ID of SGD: S000004100. The nucleotide sequence of the protein-coding region of this gene and the amino acid sequence of the encoded protein are shown in SEQ ID NOS: 1 and 2, respectively. CCW14 gene is also called YLR390W-A and SGD ID is SGD: S000006429. The nucleotide sequence of the protein-coding region of this gene and the amino acid sequence of the encoded protein are shown in SEQ ID NOS:3 and 4, respectively. The DAN1 gene is also called YJR150C and the SGD ID is SGD: S000003911. The nucleotide sequence of the protein-coding region of this gene and the amino acid sequence of the encoded protein are shown in SEQ ID NOS:5 and 6, respectively. The TIP1 gene is also called YBR067C and the SGD ID is SGD: S000000271. The nucleotide sequence of the protein-coding region of this gene and the amino acid sequence of the encoded protein are shown in SEQ ID NOS:7 and 8, respectively. The CWP1 gene is also called YKL096W and the SGD ID is SGD: S000001579. The nucleotide sequence of the protein-coding region of this gene and the amino acid sequence of the encoded protein are shown in SEQ ID NOS:9 and 10, respectively.

The “corresponding gene” is a gene that can affect the construction of the yeast cell wall and is functionally equivalent to the CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene, or CWP1 gene (e.g., different name ), which are also included as "cell wall-associated genes" in the present invention. Such "corresponding genes" include, for example, functionally equivalent genes in yeast other than Saccharomyces. Such functionally equivalent genes have, for example, the above sequence identity with the amino acid sequence of the protein encoded by each of the above genes, for example, 74% or more, 78% or more, 80% or more, 85% or more, 90% or more. % or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity. can be a gene encoding

In one embodiment, the transformed yeast of the present invention lacks the function of at least one of the CCW12 and CCW14 genes (or corresponding genes) among these genes in the host yeast. Both the CCW12 gene and the CCW14 gene may be functionally deficient. In another embodiment, the transformed yeast of the present invention lacks the function of all of the CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene and CWP1 gene (or corresponding genes) in the host yeast.

Furthermore, the host yeast may be deficient in the function of the SED1 gene (or corresponding gene) in addition to the deficient function of the above genes. The SED1 gene (Sed1) is a major cell surface-localized protein in the stationary phase of the yeast Saccharomyces cerevisiae, as described above. This gene is also called YDR077W and the SGD ID is SGD: S000002484. The nucleotide sequence of the protein-coding region of the gene for SED1 and the amino acid sequence of the encoded protein are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. A "corresponding gene" refers to a functionally equivalent gene as described above, and includes, for example, a gene encoding a protein having the above sequence identity with the amino acid sequence of SEQ ID NO:12.

Such "loss of function" of the gene in the host yeast means that the function of the target gene is lost through the artificial manipulation described later, and the host yeast originally contains the target gene itself. otherwise excluded. Loss of function of the gene can be achieved, for example, by disrupting or suppressing the expression of these genes in the host yeast. Such "deficient" embodiments include suppression of protein expression, suppression of normal protein production, and production or enhancement of non-functional mutant proteins. Genetic manipulations for this purpose include transgenics, gene knockouts, knockins, and the like. For example, gene knockout into a host cell may result in the complete deletion of that gene from the host cell. As a method of gene knockout, for example, the CRISPR-Cas9 system can be used.

Such deficient yeast may be prepared by appropriately preparing primers and the like based on known gene sequence information and carrying out the genetic manipulation described above, or commercially available deficient strains (knockout strains) may be used. Commercially available defective strains include, for example, Yeast Knockout Collection of yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) (obtained from Open Biosystems, Thermo Fisher Scientific, etc.).

"Introduction" of a polynucleotide into a host yeast means not only introduction of a polynucleotide or a gene (DNA encoding a protein of interest) for expression in an expression cassette or expression vector into a host cell, but also It also includes being expressed in The introduction method is not particularly limited, and a known method can be adopted. A typical example is a method of transforming yeast using the expression vector of the present invention described above. The transformation method is not particularly limited, and known methods such as transfection methods such as calcium phosphate method, electroporation method, lipofection method, DEAE dextran method, lithium acetate method, protoplast method, and microinjection method are used without limitation. can. The introduced gene may exist in the form of a plasmid, or may exist in the form of being inserted into the yeast chromosome or in the form of being integrated into the yeast chromosome by homologous recombination.

Yeast into which a polynucleotide has been introduced can be selected according to a conventional method, using traits by a yeast selectable marker, activity of a target protein, or the like as an index.

In addition, it can be confirmed by a conventional method that the target protein is immobilized on the cell surface of the obtained yeast (cell surface display). For example, a method in which an antibody against this protein and a fluorescence-labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase are allowed to act on the yeast to be tested; an antibody against this protein and a biotin-labeled secondary antibody; and a method in which fluorescent-labeled streptavidin is allowed to act after the reaction.

Yeast can also be transformed to express multiple types of proteins on the cell surface. In this case, each expression vector containing gene expression cassettes of sequences encoding each of a plurality of proteins may be constructed, or a plurality of gene expression cassettes may be put into one expression vector.

The transformed yeast of the present invention can be cultured under culture conditions generally applicable to yeast. Cultivation of transformed yeast can be carried out as appropriate by methods well known to those skilled in the art. The medium composition, culture pH and culture temperature can be appropriately set according to the properties of the yeast and the target protein. Cell density and culture time during culture can also be appropriately set according to the properties of the yeast and the target protein.

The transformed yeast of the present invention can be used for various purposes depending on the target protein. For example, in an embodiment in which the target protein is an enzyme, an enzymatic agent containing the transformed yeast of the present invention (enzyme surface-displaying yeast) is further provided. Examples of such an enzyme preparation include suspensions containing enzyme surface-displaying yeast and a medium capable of maintaining the surface-displaying yeast, and freeze-dried enzyme surface-displaying yeast. Such enzymatic agents can be used as biocatalysts. "Biocatalyst" is used in applications that utilize the activity of enzymes displayed on the cell surface of enzyme surface-displaying yeast, and the activity of enzymes displayed on the cell surface of enzyme surface-displaying yeast and the fermentation of the yeast itself and those used in applications that utilize both capabilities. For example, when the enzymatic agent contains yeast that expresses cellulolytic enzymes, amylolytic enzymes, etc. on the cell surface, the enzymatic agent causes decomposition (saccharification) of polysaccharides whose substrates are displayed on the surface of the enzymes and yeast fermentation. Both reactions can be catalyzed. For example, an enzymatic agent containing yeast that expresses a cellulolytic enzyme on the cell surface can be used, for example, for ethanol production using biomass. Furthermore, for example, yeast expressing lipase on the cell surface can be used as a biocatalyst for the production of biodiesel. For example, yeast that express antibodies or ligands on their cell surfaces can serve as libraries of antibodies or ligands.

(3. Method for producing ethanol)
Yeast expressing a cellulolytic enzyme according to the present invention on the cell surface can be used, for example, for the production of ethanol. Such yeasts are, for example, yeasts that display at least one enzyme selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase on the cell surface (herein, also referred to as "cellulase cell surface-displaying yeast"). is called). Alternatively, such yeast is a yeast that displays two enzymes selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase; or endoglucanase, cellobiohydrolase, and β-glucosidase. There may be.

Cellulase cell surface-displaying yeast can utilize β-1,4-linked glucose polysaccharides as fermentation substrates. In the β-1,4-linked glucose polysaccharide, a glycosidic bond (β-1,4 glycosidic bond) is formed between the 1-position of one β-glucose and the 4-position of another β-glucose that constitute the polysaccharide. ing. β-1,4-linked glucose polysaccharides include cellulose and its glycated products (eg, cellobiose, cellooligosaccharides). For β-1,4-linked glucose polysaccharides, their sources or materials include, for example, biomass. Biomass refers to industrial resources originating from substances that constitute living organisms, which are not exhaustible resources. In other words, biomass refers to renewable organic resources derived from living organisms, excluding fossil resources. Biomass may include cellulose. Biomass is not particularly limited, and examples thereof include resource crops and their wastes. The resource crops are not particularly limited, and examples thereof include corn and sugarcane, and examples of wastes of resource crops include wastes generated in the treatment process thereof. Lignocellulosic biomass is a preferred material because it does not compete with food. The lignocellulosic biomass is not particularly limited. waste generated from products consisting of

If desired, the fermentation substrate material (eg biomass) may be pretreated prior to being subjected to fermentation. Such pretreatment can break down polysaccharides (eg, cellulose) in the biomass into oligosaccharides or monosaccharides by "saccharification." The pretreatment method is not particularly limited, but includes, for example, an enzymatic method, a dilute sulfuric acid method, and a hydrothermal decomposition method. From the viewpoint of cost, the dilute sulfuric acid method and the hydrothermal decomposition method are preferred. In the dilute sulfuric acid method, for example, a material is treated with 1% (v/v) to 5% (v/v) dilute sulfuric acid at 180° C. to 200° C. for about 5 minutes to 1 hour. In the hydrothermal decomposition method, for example, materials are treated with water of about 10 MPa at 130°C to 300°C.

The cellulase cell surface-displaying yeast can be cultured under aerobic conditions before being subjected to fermentation to increase its cell mass. Cultivation of transformed yeast can be carried out as appropriate by methods well known to those skilled in the art. The pH of the medium is, for example, 4-6, preferably 5. The dissolved oxygen concentration in the medium during aerobic culture is, for example, 0.5 ppm to 6 ppm, preferably 1 ppm to 4 ppm, and more preferably 2 ppm. The culture temperature is, for example, 20°C to 45°C, preferably 25°C to 35°C, more preferably 30°C. It is preferable to culture until the amount of yeast cells is, for example, 10 g (wet weight) / L or more, preferably 12.5 g (wet weight) / L, more preferably 15 g (wet weight) / L or more, and the culture time is For example, it is about 24 hours to 96 hours.

In fermentation culture, culture conditions generally applied to yeast can be appropriately selected and used. Typically, static culture, shaking culture, aeration-stirring culture, or the like can be used for culture for fermentation. Aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. The culture temperature is, for example, 25°C to 45°C, preferably 30°C to 40°C, more preferably 35°C. Any culture time can be set as necessary, and the culture time can be set within a range of, for example, 24 hours to 120 hours. Adjustment of pH can be performed using an inorganic or organic acid, an alkaline solution, or the like. In addition to the fermentation substrate, the fermentation medium can optionally further contain medium components that can be added for culturing yeast.

After completion of ethanol fermentation, a step of recovering an ethanol-containing fraction from the culture solution (fermentation solution) and a step of further purifying or concentrating it can also be carried out. These steps and the means required for them are appropriately selected by those skilled in the art.

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

The yeast Saccharomyces cerevisiae BY4741 and BY4741sed1Δ strains used in this example were obtained from Thermo Fisher Scientific.

All PCR methods shown in this example were carried out using KOD-Plus-Neo-DNA polymerase (manufactured by Toyobo Co., Ltd.).

Gene transfer into all yeasts shown in this example was performed by the lithium acetate method.

(Preparation Example 1: Preparation of vector plasmid containing Cas9 endonuclease expression cassette)
A plasmid containing the following expression cassette X1 was prepared:
X1: TEF1 promoter + Cas9 endonuclease + CYC1 terminator

The nucleotide sequence and amino acid sequence of each component contained in the above expression cassette are as shown below:
SEQ ID NO: 13: Nucleotide sequence of promoter of TEF1 derived from Saccharomyces cerevisiae SEQ ID NO: 14: Nucleotide sequence of DNA encoding Cas9 endonuclease derived from Streptococcus pyrogenes (nuclear localization signal (SV40 NLS) derived from simian virus 40 at both ends array)
SEQ ID NO: 15: Amino acid sequence of Cas9 endonuclease derived from Streptococcus pyrogenes (including simian virus 40-derived nuclear localization signal (SV40 NLS) sequences at both ends)
SEQ ID NO: 16: nucleotide sequence of CYC1 terminator derived from Saccharomyces cerevisiae

Hereinafter, the procedure for preparing a plasmid containing the Cas9 expression cassette used in this example will be described.

The Saccharomyces cerevisiae-derived gene TEF1 was amplified by PCR using the Saccharomyces cerevisiae BY4741 strain genome as a template and the primer pair TEF1pCas9-F (SEQ ID NO: 17) and TEF1pCas9-R (SEQ ID NO: 18) to obtain the TEF1 promoter. A DNA fragment containing the region was prepared. Similarly, for the Saccharomyces cerevisiae-derived gene CYC1, a PCR method was performed using the Saccharomyces cerevisiae BY4741 strain genome as a template and a primer pair CYC1tCas9-F (SEQ ID NO: 19) and DIT1t-NcoI-R (SEQ ID NO: 20). Amplified, a DNA fragment containing the terminator region downstream of the coding region of the CYC1 gene was prepared. Furthermore, by the PCR method, using the plasmid Cas9_Base (SCIENTIFIC REPORTS, 7: 8993, DOI: 10.1038/s41598-017-08356-5) as a template, a primer pair Cas9-F (SEQ ID NO: 21) and Cas9-R (SEQ ID NO: 22 ) to prepare a DNA fragment containing the coding region of the Cas9 endonuclease gene derived from Streptococcus pyrogenes. These fragments were ligated by overlap extension PCR in the order of the TEF1 promoter region, the Cas9 endonuclease gene coding region, and the CYC1 terminator region to prepare a DNA fragment containing the expression cassette X1. Finally, this DNA fragment was transferred to XhoI and NotI-HF-treated plasmid pGK415 (low-copy plasmid vector having auxotrophic marker gene LEU2: J. Biochem. 2009; 145(6) 701-708) by In-Fusion method. and the resulting plasmid was named pCL-Cas9.

(Preparation Example 2: Preparation of vector plasmid containing guide RNA (gRNA) expression cassette)
Plasmids containing each or a plurality of the following expression cassettes X2 to X6 were prepared:
X2: SNR52 promoter + gRNA for CCW12 + gRNA scaffold + SUP4 terminator X3: SNR52 promoter + gRNA for CCW14 + gRNA scaffold + SUP4 terminator X4: SNR52 promoter + gRNA for DAN1 + gRNA scaffold + SUP4 terminator X5: SNR52 promoter + gRNA + gRNA sc for TIP1 afold + SUP4 terminator X6: SNR52 promoter + gRNA for CWP1 + gRNA scaffold + SUP4 terminator

The nucleotide sequence and amino acid sequence of each component contained in the above expression cassette are as shown below:
SEQ ID NO: 23: Nucleotide sequence of promoter of small nucleolar RNA (snoRNA) gene SNR52 derived from Saccharomyces cerevisiae SEQ ID NO: 24: Base sequence of gRNA for CCW12 SEQ ID NO: 25: Base sequence of gRNA for CCW14 SEQ ID NO: 26: DAN1 Base sequence of gRNA for SEQ ID NO: 27: Base sequence of gRNA for TIP1 SEQ ID NO: 28: gRNA for CWP1
SEQ ID NO: 29: base sequence of gRNA scaffold SEQ ID NO: 30: base sequence of terminator of tRNA gene SUP4 derived from Saccharomyces cerevisiae

The procedures for preparing various plasmids containing the guide RNA (gRNA) expression cassettes used in the Examples are described below.

By PCR method, using plasmid pGK426 (high-copy plasmid vector having auxotrophic marker gene URA3: J. Biochem. 2009; 145(6) 701-708) as a template, primer pair Guide-F1 (SEQ ID NO: 31) and Guide -R1 (SEQ ID NO: 32) was used to amplify, and the resulting DNA fragment was circularized by the In-Fusion method. Furthermore, by PCR method, this circular DNA is used as a template and amplified using a primer pair Guide-F2 (SEQ ID NO: 33) and Guide-R2 (SEQ ID NO: 34) to obtain a gRNA scaffold sequence and a tRNA gene derived from Saccharomyces cerevisiae. A vector DNA fragment containing the terminator region downstream of the SUP4 coding region was prepared. Subsequently, the Saccharomyces cerevisiae-derived low nucleolar RNA (snoRNA) gene SNR52 was subjected to PCR using the Saccharomyces cerevisiae BY4741 strain genome as a template, and the primer pair SNR52p-F (SEQ ID NO: 35) and SNR52p-R ( 36) to prepare a DNA fragment containing the SNR52 promoter region. Furthermore, using this DNA fragment as a template, a primer pair SNR52p-F (SEQ ID NO: 35) and SNR52p-R-CCW12 (SEQ ID NO: 37), SNR52p-R-CCW14 (SEQ ID NO: 38), SNR52p-R-DAN1 (SEQ ID NO: 39) ), SNR52p-R-TIP1 (SEQ ID NO: 40), or SNR52p-R-CWP1 (SEQ ID NO: 41), downstream of the SNR52 promoter region CCW12 gRNA, CCW14 gRNA, DAN1 gRNA, A DNA fragment ligated with gRNA for TIP1 or gRNA for CWP1 was prepared. These DNA fragments were ligated to the vector DNA fragments described above by the In-Fusion method, and the resulting plasmids containing expression cassettes X2, X3, X4, X5, or X6 were p2gRNA-CCW12, p2gRNA-CCW14, and p2gRNA-CCW12, respectively. We named them DAN1, p2gRNA-TIP1, and p2gRNA-CWP1.

Using the plasmid p2gRNA-CCW14 as a template, amplification was performed by PCR using a primer pair gRNA-F (SEQ ID NO: 42) and gRNA-R (SEQ ID NO: 43) to prepare a DNA fragment containing the expression cassette X3. This fragment was ligated to the SmaI-treated plasmid p2gRNA-CCW12 by the In-Fusion method, and the resulting plasmid containing the expression cassettes X2 and X3 was named p2gRNA-CCW12/CCW14.

Using the plasmid p2gRNA-CWP1 as a template, amplification was performed by PCR using a primer pair gRNA-F (SEQ ID NO: 42) and gRNA-R (SEQ ID NO: 43) to prepare a DNA fragment containing the expression cassette X6. This fragment was ligated to the SmaI-treated plasmid p2gRNA-TIP1 by the In-Fusion method, and the resulting plasmid containing the expression cassettes X5 and X6 was named p2gRNA-TIP1/CWP1.

Using the plasmid p2gRNA-DAN1 as a template, amplification was performed by PCR using a primer pair gRNA-F (SEQ ID NO: 42) and gRNA-R (SEQ ID NO: 43) to prepare a DNA fragment containing the expression cassette X4. This fragment was ligated to the SmaI-treated plasmid p2gRNA-CCW12/CCW14 by the In-Fusion method, and the resulting plasmid containing the expression cassettes X2, X3 and X4 was named p2gRNA-CCW12/CCW14/DAN1.

Using the plasmid p2gRNA-DAN1 as a template, amplification was performed by PCR using a primer pair gRNA-F (SEQ ID NO: 42) and gRNA-R (SEQ ID NO: 43) to prepare a DNA fragment containing the expression cassette X4. This fragment was ligated to the SmaI-treated plasmid p2gRNA-TIP1/CWP1 by the In-Fusion method, and the resulting plasmid containing the expression cassettes X4, X5 and X6 was named p2gRNA-TIP1/CWP1/DAN1.

(Preparation Example 3: Preparation of vector plasmid containing cell surface expression cassette)
Plasmids were prepared containing the following expression cassettes X7 and X8, respectively:
X7: SED1 promoter + SED1 secretion signal + BGL1 + SED1 anchor domain + DIT1 terminator X8: SED1 promoter + SED1 secretion signal + EGII + SED1 anchor domain + DIT1 terminator

The nucleotide sequence and amino acid sequence of each component contained in the above expression cassette are as shown below:
SEQ ID NO: 44: base sequence of DNA encoding Saccharomyces cerevisiae-derived SED1 secretory signal peptide SEQ ID NO: 45: amino acid sequence of Saccharomyces cerevisiae-derived SED1 secretory signal peptide SEQ ID NO: 46: Saccharomyces cerevisiae-derived SED1 promoter Nucleotide sequence SEQ ID NO: 47: the base sequence of the DNA of the region excluding the start codon from the coding region of the SED1 anchor protein derived from Saccharomyces cerevisiae used as the SED1 anchor domain in the examples of the present invention SEQ ID NO: 48: of the present invention Amino acid sequence of the SED1 anchor protein derived from Saccharomyces cerevisiae (but not including the initiation methionine) used as the SED1 anchor domain in the Examples SEQ ID NO: 49: base of DNA encoding Aspergillus aculatus β-glucosidase 1 (BGL1) SEQ ID NO: 50: amino acid sequence of Aspergillus aculatus β-glucosidase 1 (BGL1) SEQ ID NO: 51: base sequence of DNA encoding Trichoderma reesei endoglucanase II (EGII) SEQ ID NO: 52: Trichoderma reesei endoglucanase II (EGII) amino acid SEQ ID NO: 53: Nucleotide sequence of the terminator of DIT1 from Saccharomyces cerevisiae

The procedure for preparing the plasmid containing the cell surface expression cassette used in this example is described below.

For the Saccharomyces cerevisiae-derived gene DIT1, a primer pair DIT1t-BsrGI- Amplified using F (SEQ ID NO: 54) and DIT1t-NcoI-R (SEQ ID NO: 55), a DNA fragment containing the terminator region downstream of the coding region of the DIT1 gene was prepared. On the other hand, the vector plasmid pIEG-SSS (containing the auxotrophic marker gene HIS3 and the EG expression cassette (i.e., the SED1 promoter, the secretory signal peptide sequence of SED1, the coding region of the endoglucanase II (“EGII”) gene from Trichoderma reesei, Surface expression vector having a cassette in which the coding region of the SED1 gene and the terminator region of 445 bp downstream of the coding region of the α-agglutinin gene are arranged in this order: Biotechnology and Bioengineering, Vol.113, No.11, 2016, 2358- 2366) as a template, the terminator region of the α-agglutinin gene of this plasmid was removed by amplification using the primer pair pRS403-NcoI-F (SEQ ID NO: 56) and SED1a-BsrGI-R (SEQ ID NO: 57). A DNA fragment containing the full length was prepared, treated with BsrGI and NcoI, respectively, and ligated, and the resulting plasmid containing the expression cassette X8 was named pIEG-SSSD.

Plasmid pIBG-SSS, containing the auxotrophic marker gene HIS3 and the BGL expression cassette (i.e., the SED1 promoter, the secretory signal peptide sequence of SED1, the coding region of the β-glucosidase 1 (“BGL1”) gene from Aspergillus aculeatus, the Surface expression vector having a cassette in which the coding region and the terminator region of 445 bp downstream of the coding region of the α-agglutinin gene are arranged in this order: Biotechnology and Bioengineering, Vol.113, No.11, 2016, 2358-2366) Treated with NdeI and BsrGI, a DNA fragment containing a portion of the auxotrophic marker gene HIS3, the SED1 promoter, the secretory signal peptide sequence of SED1, the coding region of the BGL gene, and the coding region of the SED1 gene was prepared. was similarly ligated with NdeI and BsrGI-treated plasmid pIEG-SSSD, and the resulting plasmid containing expression cassette X7 was named pIBG-SSSD.

(Preparation Example 4: Preparation of gene knockout yeast)
Hereinafter, the preparation procedure via the CRISPR-Cas9 system of the gene knockout yeast used in this example will be described.

The plasmid pCL-Cas9 described in Preparation Example 1 was introduced into the yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) by the lithium acetate method. Subsequently, a transformant carrying pCL-Cas9 was obtained by selection on SD medium (supplemented with histidine, methionine, and uracil). Next, the plasmid p2gRNA-CCW12/CCW14/DAN1 described in Preparation Example 2 has homology with the coding regions of the CCW12, CCW14, and DAN1 genes, respectively, and a specific amino acid codon is replaced with a stop codon. A mixture of double-stranded oligo DNAs (SEQ ID NOS: 58, 59 and 60) having the SEQ ID NOS: 58, 59 and 60 was applied to the above transformant and introduced by the lithium acetate method. Subsequently, selection was performed on SD medium (supplemented with histidine and methionine) to obtain transformants carrying pCL-Cas9 and p2gRNA-CCW12/CCW14/DAN1. Among the resulting transformants, sequence analysis confirmed that the target codons of the CCW12, CCW14, and DAN1 genes on the genome were all replaced with stop codons. (supplemented with uracil) for several days to remove the plasmid p2gRNA-CCW12/CCW14/DAN1 from the cells. After that, strains in which no growth was confirmed in SD medium (supplemented with histidine and methionine) were selected as strains in which the CCW12, CCW14 and DAN1 genes were knocked out. This strain is called the BY-ccw12/ccw14/dan1 strain.

A double-stranded oligo DNA having a sequence homologous to the plasmid p2gRNA-TIP1/CWP1 described in Preparation Example 2 and the coding regions of the TIP1 and CWP1 genes, respectively, and having specific amino acid codons replaced with termination codons. (SEQ ID NOS: 61 and 62) were mixed and introduced into the BY-ccw12/ccw14/dan1 strain by the lithium acetate method. Subsequently, selection was performed on SD medium (supplemented with histidine and methionine) to obtain transformants carrying pCL-Cas9 and p2gRNA-TIP1/CWP1. Among the resulting transformants, sequence analysis confirmed that all the target codons of the TIP1 and CWP1 genes on the genome had been replaced with stop codons. -Cas9 and p2gRNA-TIP1/CWP1 were detached from the cells. After that, CCW12, CCW14, DAN1, and TIP1 were confirmed to show no growth on both SD medium (supplemented with histidine, methionine, and leucine) and SD medium (supplemented with histidine, methionine, and uracil). , and the strain in which the CWP1 gene was knocked out. This strain is referred to as the GPImutant strain.

(Preparation Example 5: Preparation of cell surface expressing yeast)
Procedures for preparing various cell surface-expressing yeasts are described below.

(5-1: β-Glucosidase (BGL) Surface Display Strain Hosted by 5 Gene Knockout Strain)
The plasmid pIBG-SSSD described in Preparation Example 3 was treated with NdeI and introduced into the yeast Saccharomyces cerevisiae BY4741 strain and GPImutant strain, respectively, by the lithium acetate method. Transformants were then obtained by selection on SD medium (supplemented with methionine, leucine and uracil). These transformed strains are called BY-BG-SSSD strain (BGL cell surface display strain with BY4741 strain as host) and GPIm-BGSD strain (BGL cell surface display strain with 5-gene knockout strain as host), respectively.

(5-2: Endoglucanase (EG) Surface Display Strain Hosted by 5 Gene Knockout Strain)
The plasmid pIEG-SSSD described in Preparation Example 3 was treated with NdeI and introduced into yeast Saccharomyces cerevisiae BY4741 strain and GPImutant strain, respectively, by the lithium acetate method. Transformants were then obtained by selection on SD medium (supplemented with methionine, leucine and uracil). BY-EG-SSSD strain (EG cell surface display transformant with BY4741 strain as host) and GPIm-EGSD strain (EG cell surface display transformant with 5-gene knockout strain as host), respectively. called.

Cell walls of the BY-EG-SSSD and GPIm-EGSD strains were observed with a transmission electron microscope. These electron micrographs are shown in FIG. 1 (A: BY-EG-SSSD strain and B: GPIm-EGSD strain). Disordered brush-like structures were observed on the extracellular surface of the cell wall in the GPIm-EGSD strain compared to the BY-EG-SSSD strain. There was not much difference in cell wall thickness between the two strains.

(5-3: Cell surface expression yeast strain in which CCW12 and CCW14 genes are knocked out)
The plasmid pCL-Cas9 described in Preparation Example 1 was applied to the BY-BG-SSSD strain and introduced by the lithium acetate method. Then, a transformant carrying pCL-Cas9 was obtained by selection on SD medium (supplemented with methionine and uracil). Next, the plasmid p2gRNA-CCW12/CCW14 described in Preparation Example 2 and a double strand having a sequence homologous to the coding regions of the CCW12 and CCW14 genes, and having specific amino acid codons replaced with termination codons oligo DNAs (SEQ ID NOS: 58 and 59) were mixed and applied to the above transformant, and introduced by the lithium acetate method. Then, by performing selection on SD medium (supplemented with methionine), transformants retaining pCL-Cas9 and p2gRNA-CCW12/CCW14 were obtained. Among the resulting transformants, sequence analysis confirmed that all the target codons of the CCW12 and CCW14 genes on the genome had been replaced with stop codons. ) for several days to remove the plasmids pCL-Cas9 and p2gRNA-CCW12/CCW14 from the cells. After that, yeast strains in which CCW12 and CCW14 genes were knocked out and expressed on the cell surface were confirmed to have no growth in both SD medium (supplemented with methionine and leucine) and SD medium (supplemented with methionine and uracil). selected as This strain is referred to as the ccw12/ccw14-BGSD strain.

(5-4: TIP1, CWP1, and cell surface expression yeast strain in which DAN1 genes are knocked out)
The plasmid pCL-Cas9 described in Preparation Example 1 was applied to the BY-BG-SSSD strain and introduced by the lithium acetate method. Then, a transformant carrying pCL-Cas9 was obtained by selection on SD medium (supplemented with methionine and uracil). Next, the plasmid p2gRNA-TIP1/CWP1/DAN1 described in Preparation Example 2 has homology with the coding regions of the DAN1, TIP1, and CWP1 genes, respectively, and a specific amino acid codon is replaced with a termination codon. A mixture of double-stranded oligo DNAs (SEQ ID NOS: 60, 61, and 62) having the SEQ ID NOS: 60, 61, and 62 was applied to the above transformant and introduced by the lithium acetate method. Subsequently, selection was performed on SD medium (supplemented with methionine) to obtain transformants carrying pCL-Cas9 and p2gRNA-CCW12/CCW14/DAN1. Among the resulting transformants, sequence analysis confirmed that the target codons of the TIP1, CWP1, and DAN1 genes on the genome were all replaced with stop codons. (supplemented with uracil) for several days to remove the plasmids pCL-Cas9 and p2gRNA-TIP1/CWP1/DAN1 from the cells. After that, strains in which it was confirmed that no growth was observed in both SD medium (supplemented with methionine and leucine) and SD medium (supplemented with methionine and uracil) were treated with TIP1, CWP1, and DAN1 gene knockout cell surface layers. It was selected as an expression yeast strain. This strain is called tip1/cwp1/dan1-BGSD strain.

(5-5: 6 gene knockout cell surface expression yeast strain)
By PCR method, Saccharomyces cerevisiae BY4741 sed1Δ strain genome (Biotechniques.2011, 50(5):325-328) was used as a template, and a primer pair sed1-F (SEQ ID NO: 63) and sed1-R ( 64) to prepare a DNA fragment containing, in this order, a 520 bp region upstream of the coding region of the SED1 gene, the kanMX gene of the antibiotic G418 resistance marker gene, and a 526 bp region downstream of the coding region of the SED1 gene. This DNA fragment was applied to the GPIm-BGSD strain and introduced by the lithium acetate method. Transformants were then obtained by selection on SD medium (supplemented with methionine, leucine, uracil, and G418). This transformed strain is called GPIm-sed1-BGSD strain.

(Test Example 1: Examination of β-glucosidase (BGL) activity)
The β-glucosidase (BGL) activity of yeast cells (active amount (U) per dry cell weight) was examined according to the following procedure.

Cells were transferred to 5 mL of SD medium (supplemented with leucine, methionine, and uracil), cultured at 30° C. and 180 rpm for 18 hours (preculture), and then transferred to 50 mL of 1×YPD medium (initial OD ₆₀₀ =0 .05) and cultured at 30° C. and 150 rpm (main culture). The culture medium was collected every 24 hours from the start of the main culture, and centrifuged at 1,000 g for 5 minutes to separate the cells and the medium.

The β-glucosidase activity of the cells was measured as follows:
(1) washing the cells twice with distilled water;
(2) 500 μL reaction solution (composition: 100 μL 10 mM pNPG (p-nitrophenyl-β-D-glucopyranoside) (final concentration 2 mM); 500 μL sodium citrate buffer (pH 5.0) 50 μL (final concentration 50 mM); distilled water) 250 μL; and 100 μL yeast suspension) (final cell concentration 1-10 g wet cells/L)) and react at 500 rpm, 30° C. for 10 minutes;
(3) After completion of the reaction, 500 μL of 3M Na ₂ CO ₃ was added to stop the reaction; and (4) After centrifugation at 10,000 g for 5 minutes, the absorbance ABS ₄₀₀ of the supernatant at 400 nm was measured. The amount of enzyme that liberates 1 μmol of pNP (p-nitrophenol) per minute is defined as 1 U.

(Test Example 2: Examination of endoglucanase (EG) activity)
The endoglucanase (EG) activity of yeast cells (active amount per dry cell weight) was examined according to the following procedure.

Cells were transferred to 5 mL of SD medium (supplemented with leucine, methionine, and uracil), cultured at 30° C. and 180 rpm for 18 hours (preculture), and then transferred to 50 mL of 1×YPD medium (initial OD ₆₀₀ =0 .05) and cultured at 30° C. and 150 rpm (main culture). After 48 hours from the start of the main culture, each culture solution was collected and centrifuged at 1,000 g for 5 minutes to separate the cells and the medium.

Measurement of bacterial endoglucanase activity was performed as follows:
(1) washing the cells twice with distilled water;
(2) 2500 μL reaction solution (composition: 1 Cerazyme C tablet (manufactured by Megazyme); 250 μL 500 mM sodium citrate buffer (pH 5.0) (final concentration 50 mM); 2000 μL distilled water; and 250 μL yeast suspension (final Prepare a bacterial cell concentration of 10 g wet bacterial cells / L)), stand, and react at 38 ° C. for 3 hours;
(3) After completion of the reaction, centrifuge at 10,000 g for 5 minutes and measure the absorbance ABS ₅₉₀ at 590 nm of the supernatant.

(Test Example 3: Examination of ethanol production ability from cellobiose using β-glucosidase surface-displaying yeast)
β-Glucosidase (BGL) surface-displaying yeast was used to examine the ability to produce ethanol from cellobiose according to the following procedure.

Cells were transferred to 5 mL of SD medium (supplemented with leucine, methionine, and uracil), cultured at 30° C. and 180 rpm for 18 hours (preculture), and then transferred to 500 mL of 1×YPD medium (initial OD ₆₀₀ =0 .05) and cultured at 30° C. and 150 rpm (main culture). After 48 hours from the start of the main culture, each culture solution was collected, and the cells were washed twice with distilled water and used for the ethanol production test.

Ethanol production tests were conducted under the following conditions:
Cellobiose 20g/L
Yeast extract 10g/L
Peptone 20g/L
Yeast cell 10 g wet cell weight / L
Total 20mL
The above was placed in a rotary fermentation apparatus and reacted at 30° C. and 150 rpm for 8 hours without adding a commercially available enzyme agent.

(Example 1: Comparison of host yeast in β-glucosidase (BGL) cell surface display)
In this example, cell surface display transformed yeast (BY-BG-SSSD strain and GPIm-BGSD strain) obtained by introducing a plasmid containing expression cassette X7 using Saccharomyces cerevisiae BY4741 strain and its 5-gene knockout strain as hosts : prepared in 5-1 of Preparation Example 5), the β-glucosidase (BGL) activity of the cells was measured.

This result is shown in FIG. (A) β-glucosidase activity and (B) cell growth over time. In both FIGS. 2 (A) and (B), the horizontal axis indicates the culture time (“time (hour)”), and the vertical axis indicates the β-glucosidase activity (activity amount per dry cell weight (“U /g dry cell weight"), and (B) shows cell growth ( _OD600 ).The symbols in Figure 2 are as follows: black circles, strain BY-BG-SSSD; and black squares, GPIm- BGSD strain.

As shown in FIG. 2, the BGL cell surface display transformant (GPIm-BGSD strain) using a 5-gene knockout strain as a host is a transformant (BY-BG-SSSD strain) using the conventional BY4741 strain as a host. strain), it was found to exhibit considerably higher surface layer BGL activity.

(Example 2: Comparison of host yeast in endoglucanase (EG) cell surface display)
In this example, cell surface display transformed yeast (BY-EG-SSSD strain and GPIm-EGSD strain) obtained by introducing a plasmid containing expression cassette X8 using Saccharomyces cerevisiae BY4741 strain and its 5-gene knockout strain as hosts : prepared in 5-2 of Preparation Example 5), the endoglucanase (EG) activity of the cells was measured.

The results are shown in FIG. The vertical axis in FIG. 3 indicates EG relative activity (absorbance measurements (“ABS ₅₉₀ ”). The bar graphs in FIG. 3 represent, from left to right: BY-EG-SSSD and GPIm-EGSD strains.

As shown in FIG. 3, even when EG activity was used as an index, the EG cell surface display transformant (GPIm-EGSD strain) using the 5-gene knockout strain as the host was the conventional BY4741 strain as the host. It was observed to exhibit higher cell surface EG activity than the transformed strain (BY-EG-SSSD strain).

(Example 3: Examination of combinations of genes to be knocked out)
In this example, Saccharomyces cerevisiae strain BY4741, a 5-gene knockout strain, and a strain in which only two (CCW12 and CCW14) or three (TIP1, CWP1, and DAN1) of the five genes were knocked out were used as hosts for expression cassettes. Surface display transformed yeast obtained by introducing a plasmid containing X1 (BY-BG-SSSD strain, GPIm-BGSD strain (5-1 above), ccw12/ccw14-BGSD strain (5-3 above), tip1/ With regard to the cwp1/dan1-BGSD strain (above 5-4)), the BGL activity of the cells was measured.

The results are shown in FIG. (A) β-glucosidase activity and (B) cell growth over time. 4 (A) and (B), the horizontal axis indicates the culture time (“time (hour)”), and the vertical axis indicates the β-glucosidase activity (activity amount per dry cell weight (“U /g dry cell weight"), and (B) shows cell growth ( _OD600 ).The symbols in Figure 4 are as follows: black circles, strain BY-BG-SSSD; and black squares, GPIm- BGSD strains: filled diamonds, ccw12/ccw14-BGSD strains; and filled triangles, tip1/cwp1/dan1-BGSD strains.

As shown in FIG. 4, the BGL cell surface display transformant strain (ccw12/ccw14-BGSD strain) using a strain in which only CCW12 and CCW14 are knocked out as a host is a transformant strain using a 5-gene knockout strain as a host. (GPIm-BGSD strain), it was found to exhibit high cell surface BGL activity, which is almost equivalent to that of (GPIm-BGSD strain). In addition, the transformed strain (tip1/cwp1/dan1-BGSD strain) using a strain in which only TIP1, CWP1, and DAN1 were knocked out as a host was also transformed using the conventional BY4741 strain as a host (BY-BG -SSSD strain) was observed to exhibit slightly higher surface BGL activity.

(Example 4: Comparison of host yeast to which a knockout gene was added)
In this example, a cell surface display transformed yeast (GPIm-BGSD strain) (5-1 above) obtained by introducing a plasmid containing an expression cassette X7 into a 5-gene knockout strain as a host and the SED1 gene of the strain were used. With regard to the additionally knocked out transformed strain (GPIm-sed1-BGSD strain) (5-5 above), the BGL activity of the cells was measured.

The results are shown in FIG. (A) β-glucosidase activity and (B) cell growth over time. In both FIGS. 5 (A) and (B), the horizontal axis indicates the culture time (“time (hour)”), and the vertical axis indicates the β-glucosidase activity (activity amount per dry cell weight (“U /g dry cell weight"), and (B) shows cell growth (OD ₆₀₀ ). Symbols in Fig. 5 are as follows: black squares, strain GPIm-BGSD; and black diamonds, GPIm-sed1. -BGSD strain.

As shown in FIG. 5, a transformant strain (GPIm-sed1- BGSD strain) was found to exhibit high cell surface BGL activity after 72 hours of culture.

(Example 5: Comparison of host yeast in ethanol production from cellobiose using β-glucosidase cell surface display yeast)
In this example, Saccharomyces cerevisiae BY4741 strain, CCW12 and CCW14 two-gene knockout strains, CCW12, CCW14, TIP1, CWP1, and DAN1 five-gene knockout strains were used as hosts, and plasmids containing expression cassette X7 were introduced. For cell surface display transformed yeast (BY-BG-SSSD strain, ccw12/ccw14-BGSD strain, and GPIm-BGSD strain) (5-1, 5-3 above), cellobiose, which is a polymer of two molecules of glucose, compared the ethanol production capacity of

The results are shown in FIG. In both FIGS. 6A and 6B, the horizontal axis indicates the fermentation time (“time (hour)”), and the vertical axis indicates the cellobiose concentration (g/L) in (A) and the ethanol concentration (g/L) in (B). /L). Symbols in FIG. 6 are as follows: filled circles, BY-BG-SSSD strain; filled diamonds, ccw12/ccw14-BGSD strain; and filled squares, GPIm-BGSD strain.

As shown in FIG. 6, a BGL cell surface display transformant (ccw12/ccw14-BGSD strain) using a 2-gene knockout strain as a host and a BGL cell surface display transformant (ccw12/ccw14-BGSD strain) using a 5-gene knockout strain as a host ( GPIm-BGSD strain) was confirmed to produce ethanol from cellobiose at a faster rate than the conventional transformed strain using BY4741 strain as a host (BY-EG-SSSD strain).

INDUSTRIAL APPLICABILITY According to the present invention, it is extremely useful for improving the efficiency of substance production using yeast, reducing costs, and promoting its spread. As a more specific application field, it is possible to improve the efficiency and reduce the cost of chemical production from cellulosic biomass by yeast that presents cellulolytic enzymes on the cell surface. Furthermore, for example, the efficiency of biodiesel production using yeast displaying lipase on the cell surface, and the use of yeast libraries displaying antibodies or ligands on the cell surface for drug screening are conceivable.

Claims (10)

A transformed yeast that expresses a protein on the cell surface,
Amino acids having a sequence identity of 90% or more with the amino acid sequences of at least the CCW12 gene, the CCW14 gene, and the proteins encoded by these genes among the cell wall-related genes in the host yeast harboring the cell wall-related genes The sequence lacks the function of a gene encoding a protein , and a polynucleotide containing a promoter, a secretory signal sequence, a DNA encoding the protein, a DNA encoding an anchor domain and a terminator has been introduced.
transformed yeast. 2. The transformed yeast according to claim 1, wherein the functions of all of the cell wall-associated genes of CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene and CWP1 gene are deficient. 3. The transformed yeast according to claim 1, further lacking the function of the SED1 gene. 4. The transformed yeast according to any one of claims 1 to 3 , wherein said protein is an enzyme. 5. The transformed yeast of claim 4 , wherein said enzyme is a cellulolytic enzyme. The transformed yeast according to any one of claims 1 to 5 , wherein the host yeast is a yeast belonging to the genus Saccharomyces. A method for producing a transformed yeast that expresses a protein on the cell surface, comprising:
Amino acids having a sequence identity of 90% or more with the amino acid sequences of at least the CCW12 gene, the CCW14 gene, and the proteins encoded by these genes among the cell wall-related genes in the host yeast harboring the cell wall-related genes Deactivating the gene encoding the protein, which is the sequence, and introducing into the host yeast a polynucleotide comprising a promoter, a secretory signal sequence, DNA encoding the protein, DNA encoding the anchor domain and a terminator. A method, including An enzymatic agent comprising the transformed yeast according to claim 4 or 5 . The enzymatic agent according to claim 8 , which is used for ethanol production using biomass. A method for producing ethanol,
A method comprising culturing the transformed yeast according to claim 5 in a medium containing β-1,4-linked glucose polysaccharides.

Images