JP2005245335A - Target protein-expressing yeast and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a yeast that has a target protein on the cell cortex and can express the target protein in high efficiency under the industrial environment. <P>SOLUTION: In this practical yeast, from the 5'-terminus in order, a high active promoter selected from the group consisting of yeast SED1 promoter, GAPDH promoter, PGK1 promoter and ADH1 promoter, secretion signal that functions in the yeast cells, the sequence encoding the target protein and a chromosome integration type expression vector that has a sequence encoding the plasma membrane bound area of the GPI anchoring protein are incorporated into the chromosomal DNA. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a yeast that retains a useful protein on the cell surface, an expression vector for producing such yeast, and a method for producing a polysaccharide derived from plant biomass such as glucose and xylan degradation products using this yeast. About.

  In recent years, production of useful proteins using microorganisms such as E. coli and yeast that have been genetically modified has been industrially performed. As protein production methods using microorganisms, there are two general methods: a method in which a target protein is accumulated in a recombinant cell and a method in which the target protein is secreted outside the cell.

  When the target protein is an enzyme, the target substance is produced by allowing the enzyme extracted from the recombinant to act on the substrate, or the recombinant that produces the enzyme is allowed to act on the substrate. Is done. The method using an isolated and purified enzyme is complicated in extraction operation and has a low enzyme recovery rate, which increases the cost and is not suitable for industrial use. On the other hand, the method for producing a target substance from a substrate using a recombinant that produces an enzyme has the following drawbacks.

  When a recombinant that accumulates the enzyme, which is the target protein, in the microbial cells is used, permeation of the substrate into the cell membrane becomes the rate-determining enzyme reaction. That is, in order for the enzyme reaction to be carried out in the microbial cells, it is necessary for the substrate to permeate the cell membrane and enter the cell, so a high molecular substrate that does not permeate the cell membrane cannot be used for the reaction. Or the reaction rate becomes slow. In addition, depending on the type of protein to be produced, the growth of the host may be inhibited, and there are certain limitations to the application of this method.

  On the other hand, when using recombinants that secrete the target protein outside the cells, the enzyme concentration in the reaction system is generally low, and even if a sufficient amount of product is obtained at the test tube level, There is a tendency that a practically sufficient amount of product cannot be obtained. In addition, the enzyme may be degraded by the action of a proteolytic enzyme such as a protease present in the reaction system.

  As a method for solving the above-mentioned problems, “yeast cell surface display engineering” in which a target protein such as an enzyme is displayed on the cell surface of yeast has attracted attention. This is a technique for presenting a target protein at a high density on the cell surface of yeast by expressing the target protein as a fusion protein with a protein that can be immobilized on the cell surface, such as a GPI anchoring protein.

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

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

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

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

  Such “yeast cell surface display engineering” is a new research field, and various studies are currently being conducted at the laboratory level. At present, as a research stage, an autonomous propagation vector, a glycolytic enzyme promoter, and laboratory yeast, which are general genetic manipulation materials, are used. In detail, a self-replicating vector in which a sequence encoding a target protein downstream of a glycolytic promoter and a sequence encoding a part of a cell surface localized protein such as a GPI anchoring protein are linked to a laboratory. By performing yeast transformation, the target protein is immobilized on the cell surface of the yeast.

  However, the yeast cell surface display system using these materials has the following drawbacks in industrial material production.

  As the autonomously proliferating vector, a multicopy autonomously replicating plasmid vector that can exist in one cell is usually used. In the autonomously growing plasmid vector, the plasmid drops off as the cell growth progresses. Therefore, in the laboratory, dropping of the plasmid is prevented by applying a selective pressure using a medium containing a substance corresponding to the selection marker. However, it is not practical to culture yeast while applying selective pressure continuously in industrial production because it takes time and costs.

  Laboratory yeast is a strain that has been bred mainly for research purposes, and is generally given many auxotrophy. For this reason, when using a medium containing various required nutrients and culturing at a constant temperature of about 30 ° C. while maintaining the medium pH at about 7 while sufficiently supplying oxygen, a sufficient growth rate is obtained. However, in an industrial environment that does not satisfy these conditions, a practically sufficient growth rate cannot be obtained.

  The present invention relates to a yeast in which a target protein is immobilized on a cell surface, which can produce the target protein with high efficiency even in an industrial environment, a polynucleotide and an expression vector for producing the yeast, and the yeast. It is an object of the present invention to provide a method for efficiently producing a target substance.

In order to solve the above-mentioned problems, the present inventor repeated research and obtained the following knowledge.
(1) From the 5 'end side, a specific highly active promoter and a secretory signal sequence that functions in yeast cells and is linked so that it can be expressed, a sequence encoding the target protein, and a part of the cell surface localized protein Yeast in which an expression vector or polynucleotide comprising a sequence encoding a GPI and a unit comprising a GPI anchor attachment signal sequence is incorporated into chromosomal DNA uses a specific highly active promoter, so even under a stress environment The target protein can be stably highly expressed. In addition, since the sequence encoding the target protein is integrated into the chromosome, the target protein can be stably produced with high efficiency without applying selective pressure.
(2) In addition, when the yeast in which the above expression vector is incorporated into the chromosomal DNA is a practical yeast, it can be easily grown in a stress environment. Moreover, since practical yeast shows high tolerance to ethanol, after production of monosaccharides, the entire reaction system can be directly subjected to ethanol fermentation.

  The present invention has been completed based on the above findings, and provides the following polynucleotide, expression vector, yeast, and a method for producing glucose and a method for producing a xylan degradation product using this yeast.

  Item 1. In order from the 5 ′ end, a highly active promoter selected from the group consisting of the yeast SED1 promoter, GAPDH promoter, PGK1 promoter, and ADH1 promoter, a secretory signal sequence that functions in yeast cells, a sequence encoding the target protein, and a cell surface localized protein A polynucleotide comprising a sequence encoding a portion of the GPI anchor attachment signal sequence.

  Item 2. Item 2. The polynucleotide according to Item 1, wherein the highly active promoter is a yeast SED1 promoter.

  Item 3. A chromosome integration type expression vector comprising the polynucleotide according to Item 1 or 2.

  Item 4. A yeast in which the polynucleotide of Item 1 or 2 or the expression vector of Item 3 is incorporated into chromosomal DNA.

  Item 5. Item 5. The yeast according to Item 4, which is a practical yeast.

  Item 6. Item 6. The method for producing monosaccharides comprising the step of decomposing a polysaccharide derived from plant biomass by the presence of the yeast according to item 4 or 5, wherein the target protein is a sugar hydrolase, and the step of recovering the monosaccharide to be produced .

  Item 7. Item 6. A method for producing glucose, comprising a step of degrading starch in the presence of the yeast according to Item 4 or 5, wherein the target protein is amylase, and a step of recovering the produced glucose.

  Item 8. Item 8. The method according to Item 7, wherein the amylase is glucoamylase.

  Item 9. Item 6. The yeast according to Item 4 or 5, wherein the target protein is arabinofuranosidase, and a xylan degradation product comprising a step of decomposing xylan in the presence of xylosidase or further xylanase, and a step of recovering the produced xylan degradation product. Manufacturing method.

  Item 10. The step (a) of degrading xylan in the presence of the yeast according to item 4 or 5, wherein the target protein is arabinofuranosidase, and the xylan partial degradation product obtained in step (a) is converted into xylosidase or further xylanase A method for producing a xylan decomposition product, comprising the step (b) of decomposing in the presence, and the step of recovering the generated xylan decomposition product.

  Since the polynucleotide and the expression vector of the present invention use a specific highly active promoter, the target protein can be stably highly expressed even under industrial scale culture conditions.

  In addition, since the expression vector of the present invention is a host chromosome integration type vector, the expression unit is not easily dropped without applying selective pressure. For this reason, the target protein can be highly expressed even in culture on an industrial scale where it is difficult to apply selective pressure in terms of cost. Similar effects can be obtained when the polynucleotide of the present invention is directly integrated into the host chromosome.

  Furthermore, by introducing the expression vector or polynucleotide of the present invention into a practical yeast, a high growth rate can be obtained even in cultivation on an industrial scale, which may result in severe culture conditions. In addition, since practical yeasts are polyploid, it is possible to incorporate a plurality of the expression vectors of the present invention into homologous chromosomes, and as a result, the target protein is compared with the case of integrating into laboratory yeasts that are often haploid. The expression level of becomes higher. Furthermore, since practical yeasts have extremely high resistance to ethanol, they can be directly subjected to ethanol fermentation after producing monosaccharides.

Hereinafter, the present invention will be described in detail.
(I) Polynucleotide The polynucleotide of the present invention comprises, from the 5 ′ end, a highly active promoter selected from the group consisting of a yeast SED1 promoter, a GAPDH promoter, a PGK1 promoter, and an ADH1 promoter, a secretion signal that functions in yeast cells. A polynucleotide (ie, an expression unit) comprising a sequence, a sequence encoding a protein of interest, a portion of a cell surface localized protein, and a GPI anchor attachment signal sequence.

  A secretory signal sequence, a sequence encoding a target protein, a part of a cell surface localized protein, and a fragment comprising a GPI anchor attachment signal sequence are linked to a position where it can be expressed by a highly active promoter.

The polynucleotide may be inserted into a chromosome integration type expression vector. The structure of this expression unit inserted into a chromosomally integrated expression vector is shown in FIG.
Highly active promoter In the present invention, a specific highly active promoter is used. Specific high activity promoters include yeast SED1 promoter (Japanese Patent Laid-Open No. 2003-265177), GAPDH promoter (Japanese Patent Publication No. 07-24594), PGK1 promoter (EMBO J. (1982), 1,603- Tuite, MF et al), and ADH1 It is a promoter selected from the group consisting of promoters (Nature (1981), 293, 717-Hitzeman, RA et al). These promoters exhibit high promoter activity in yeast cells.

  Among others, the present inventors have proved that the SED1 promoter, which is a promoter of the SED1 gene encoding a yeast cell wall protein, exhibits a very strong promoter activity (Japanese Patent Laid-Open No. 2003-265177).

  The yeast SED1 promoter not only exhibits high transcriptional activity under normal culture conditions, but also exhibits stable high promoter activity even under various stress environments such as high temperature, high osmotic pressure or low osmotic pressure, oligotrophic conditions, and the presence of alcohol. Show. In addition, it is not easily affected by changes in pH.

  When culturing on an industrial scale, a reaction system in which various components are dissolved, that is, a high osmotic pressure environment is often obtained. In addition, it is difficult to strictly control the temperature and pH compared to the case of culturing in a laboratory, and it is difficult to use a nutrient rich culture medium in terms of cost and the like. Thus, since the culture environment on an industrial scale is a stress environment for yeast, the polynucleotide or expression vector containing the specific highly active promoter can be suitably used for industrial substance production using recombinant yeast. It becomes.

The specific high activity promoter may be the entire region or a partial region as long as it exhibits high promoter activity.
Secretory signal sequence The secretory signal sequence contained in the polynucleotide of the present invention is a polynucleotide sequence encoding a secretory signal peptide.

  A secretory signal peptide is a peptide that is normally bound to the N-terminus of a secreted protein that is secreted extracellularly, including the periplasm. This peptide has a similar structure between organisms, consists of about 20 amino acids, has a basic amino acid sequence in the vicinity of the N-terminus, and then contains many hydrophobic amino acids. The secretory signal is usually removed by degradation by a signal peptidase when the secreted protein is secreted from inside the cell through the cell membrane to the outside of the cell.

In the present invention, any polynucleotide sequence encoding a secretory signal peptide capable of secreting arabinofuranosidase outside the yeast can be used, and the origin thereof is not limited. For example, secretion signal peptide sequence of glucoamylase such as Rhizopus oryzae, secretion signal peptide sequence of glucoamylase of Aspergillus oryzae, secretion signal of α- or a-agglutinin of yeast (Saccharomyces serevisiae) Peptide sequences, secretory signal peptide sequences of α-factor derived from yeast (Saccharomyces serevisiae), and the like can be suitably used. In particular, the secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase is preferable from the viewpoint of secretion efficiency. In addition, a secretory signal peptide sequence of Aspergillus oryzae-derived glucoamylase is also preferred.
The kind of the sequence target protein encoding the target protein is not particularly limited. Since the object of the present invention is mainly the production of monosaccharides serving as a substrate for ethanol fermentation, preferred examples of the target protein include glycolytic enzymes. Examples of glycolytic enzymes include exo-1,4-α-D-glucosidase (glucoamylase), β-amylase, exoisomaltotriohydrolase, exomaltotetraohydrolase, exomaltohexohydrolase, and α-glucosidase. Exo-type amylase (starch hydrolase); endo-type amylase (starch hydrolase) such as α-amylase, pullulanase, isoamylase; cellulose degradation such as endo-1,4-β-glucanase (cellulase) Enzymes; xylan degrading enzymes such as arabinofuranosidase, xylanase, and xylosidase can be exemplified.

  Among them, amylase can be preferably used since it exhibits high degradation activity. Among amylases, glucoamylase is more preferable in that starch can be decomposed completely or almost completely to glucose because not only α1 → 4 glucoside bond but also α1 → 6 glucoside bond can be decomposed.

  These enzymes may be derived from any organism such as bacteria, fungi, plants and animals. Polynucleotides encoding these enzymes can be easily obtained by those skilled in the art. For example, a polynucleotide encoding Aspergillus oryzae glucoamylase is exemplified by the nucleotide sequence described in JP-A-10-84968. It can be easily obtained by PCR using primers designed based on the above.

The polynucleotide sequence encoding the target protein may be a sequence encoding the entire length thereof, or may be a sequence encoding a partial region of the target protein as long as it exhibits the activity of the target protein. Moreover, as long as the activity of the target protein is exhibited, the sequence encoding the natural protein may be a sequence in which one or more nucleotides are deleted, added or substituted.
Cell surface localized protein
<Cell surface localized protein>
In the method of the present invention, any protein may be used as long as it is a protein that is immobilized on, adhered to, or adhered to the cell surface and is localized there, and any known protein can be used without limitation.

Examples of proteins localized in the cell membrane include transmembrane proteins and cell surface localized proteins. The transmembrane protein is a protein that penetrates the lipid bilayer at the hydrophobic amino acid region, and is often found in receptor proteins. On the other hand, a protein modified with a lipid is known as a cell surface localized protein, and this lipid is immobilized on a cell membrane by covalently binding to a membrane component. In addition, there are cell surface localized proteins whose mechanism of immobilization has not been clarified, and these can be used in the method of the present invention. As cell surface localized proteins, various GPI anchoring proteins and BGL2 described later are known. BGL2 is known to bind strongly to the cell wall with yeast β-glucanase, but its mechanism is unknown. BGL2 does not have a motif common to GPI anchoring proteins.
<GPI anchoring protein>
As a representative example of cell surface localized protein, GPI (glycosylphosphatidylinositol: Glycolipid based on ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid) anchoring protein Can be mentioned. GPI anchoring protein has GPI which is a glycolipid at its C-terminal, and this GPI is bound to the cell membrane surface by covalently binding to PI (phosphatidylinositol) in the cell membrane.

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

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

FIG. 2 shows the processing of GPI anchoring protein and intracellular transport.
<GPI anchoring protein as one example of cell surface localized protein of the present invention>
Thus, the GPI anchoring protein is one of the cell surface localized proteins referred to in the present invention and has a GPI anchor adhesion signal. In the present invention, a polynucleotide encoding a cell membrane binding region of a GPI anchoring protein containing a GPI anchor attachment signal region is preferably used as a sequence consisting of a part of a cell surface localized protein and a GPI anchor attachment signal sequence. Can be used.

  The cell membrane binding region of the GPI anchoring protein is usually a C-terminal region including a GPI anchor attachment signal region. This cell membrane-binding region only needs to contain a GPI anchor attachment signal region, and may contain any part of other GPI anchoring protein as long as it does not inhibit arabinofuranosidase activity.

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

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

A sequence encoding a cell membrane binding region of a GPI anchoring protein such as α-agglutinin inherently contains both a sequence encoding a part of a cell surface localized protein and a GPI anchor attachment signal sequence. In the present invention, the sequence encoding a part of the cell surface localized protein and the GPI anchor attachment signal sequence may be prepared from different sources.
Expression Vector As described above, the polynucleotide of the present invention may be incorporated into a chromosomally integrated expression vector. An expression vector is an expression vector that is incorporated into its chromosomal DNA when introduced into yeast. As long as the expression vector does not contain an autonomously replicating sequence of yeast, it is usually a yeast chromosomal DNA integration type.

  The expression vector includes a highly active promoter and a polynucleotide fragment operably linked thereto, the polynucleotide fragment comprising the secretory signal sequence, a target protein coding sequence, a part of a cell surface localized protein, and Has a GPI anchor attachment signal sequence.

  Any oligonucleotide sequence may be inserted between the secretory signal sequence and the target protein coding sequence as long as the secretion of the fusion protein by the secretory signal peptide is not inhibited. In general, insertion sequences up to about 30b are allowed. Further, an arbitrary oligonucleotide sequence may be inserted between the target protein coding sequence and the sequence encoding the cell membrane binding region of the GPI anchoring protein as long as it is generally up to about 30b. In addition, the expression of this unit by the promoter is also inhibited between the highly active promoter and the polynucleotide fragment comprising the secretory signal sequence, the target protein coding sequence, part of the cell surface localized protein, and the GPI anchor attachment signal sequence. Any nucleotide sequence may be inserted as long as it is not.

  The expression vector may be a plasmid vector or an artificial chromosome. A plasmid vector is preferable in that the preparation of the vector is easy and the transformation of yeast cells is easy.

  It is desirable that the expression vector has an E. coli replication origin (ColE1) so that the expression vector can be easily constructed using E. coli. In addition, it is usually sufficient to have a yeast selection marker (for example, drug resistance gene, URA3, TRP, LEU2, etc.) and an E. coli selection marker (drug resistance gene, etc.). In addition to the SED1 promoter, an operator, an enhancer, a terminator, etc. may be included in order to regulate the expression of the target gene. Examples of the terminator include ADH1 (aldehyde dehydrogenase) terminator, GAPDH (glyceraldehyde 3′-phosphate dehydrogenase) terminator, and the like.

  An expression vector having a SED1 promoter, which is incorporated into yeast chromosomal DNA, is a region consisting of 320 amino acids from the C-terminal of pRS406, SED1 promoter and secretory signal sequence, structural protein sequence of target protein and α-agglutinin PRS406 Psed1-CAS1 (see Examples in the present specification) constructed by inserting a polynucleotide fragment consisting of a sequence coding for.

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

(II) Yeast The yeast of the present invention is a yeast that holds the polynucleotide or expression vector of the present invention, and preferably the polynucleotide or expression vector is integrated into the chromosomal DNA.

  The yeast of the present invention can be obtained by introducing the polynucleotide or expression vector of the present invention into a yeast serving as a host. 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. it can.

  Yeast into which the polynucleotide of the present invention has been introduced can be selected according to a conventional method using, for example, a trait by a selection marker such as URA3 or the activity of the target protein. A transformant in which a plurality of expression units derived from an expression vector are introduced can be easily obtained by selecting one having a high activity using the activity of the target protein as an index. When a drug resistance gene is used as a selection marker, a transformant having a plurality of expression vectors introduced therein can be easily selected by selecting at a drug concentration that is changed stepwise or gradually. For example, when KanMX4 is used as a selection marker, a transformant introduced with a higher copy number expression vector can be selected by using a medium having a gradient or a step in the geneticin concentration.

It can be confirmed by a conventional method that the target protein is immobilized on the cell surface layer of the obtained yeast. For example, a method in which an antibody against this protein and a fluorescently labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase are allowed to act on a test yeast, an antibody against this protein and a biotin-labeled secondary antibody And a method in which a fluorescent labeled streptavidin is allowed to act after the reaction.
<Host>
The yeast used as the host is not particularly limited as long as it belongs to Ascomycetou yeast. Among them, those belonging to Saccharomycetaceae are preferable, and those belonging to Saccharomyces are more preferable.

  Among these, practical yeasts are preferable because they are resistant to various culture stresses and exhibit stable cell growth even in industrial production in which strict control is difficult. Practical yeasts include yeasts that are deeply involved in fermented foods, such as sake yeast, shochu yeast, wine yeast, beer yeast, and baker's yeast. Among practical yeasts, sake yeast having high fermentability and high ethanol tolerance and genetically stable is preferable.

  In general, ethanol is toxic to microorganisms. Yeast that undergoes ethanol fermentation is no exception, and when the ethanol concentration exceeds 8% (v / v), it is gradually killed by the ethanol it produces. Here, sake yeast is a strain that has been selected and bred for many years with sake moromi with an ethanol concentration as high as 20%, and has extremely high ethanol resistance compared to general yeast.

  In the present invention, xylose and arabinose are produced by decomposition of xylan using yeast, and these pentoses are used as an ethanol fermentation raw material. In ethanol fermentation, since it is important to increase the ethanol concentration of the system to the ultimate, the yeast used therein is required to have high ethanol resistance.

Examples of sake yeast include “Kyokai Yeast” distributed by Japan Brewing Association and mutant strains using these as parent strains. In addition, any yeast that is used in sake brewing is useful. When an auxotrophic gene is used as a transformation marker, a strain obtained by imparting auxotrophy to these sake yeasts using a technique such as mutation may be used. Such auxotrophs include uracil auxotrophy and tryptophan. Requirement, leucine requirement, histidine requirement, adenine requirement and the like. Saccharomyces cerevisiae GRI-117-U obtained by a mutation method using “Kyokai No. 9 yeast” as a host is given as an example of imparting uracil requirement to sake yeast.
(III) Method for Producing Glucose The method for producing glucose of the present invention includes a step of decomposing starch in the presence of the yeast of the present invention and a step of recovering the produced glucose. As yeast, one in which a polynucleotide encoding amylase as a target protein is incorporated into chromosomal DNA is used. Amylase refers to starch hydrolase. One type of yeast can be used alone, or different types of yeasts producing different types of amylases can be used in combination. It is also possible to use a yeast that has produced a plurality of types of amylases by incorporating polynucleotides encoding a plurality of types of amylases into the chromosomal DNA.

When glucoamylase is used as amylase, starch can be decomposed into glucose by using one kind of yeast that produces amylase. In addition, for example, by using a yeast that produces endo-type α-amylase that degrades α1 → 4 glucoside bonds and a yeast that produces endo-type pullulanase or isoamylase that degrades α1 → 6 glucoside bonds. Can also produce glucose from starch. Furthermore, starch can be liquefied first using yeast that produces α-amylase, and then glucose can be produced using yeast that produces glucoamylase and yeast that produces pullulanase.
As the starch raw material starch, easily derived plant-derived starch can be suitably used. For example, starch may be extracted from grains such as rice, wheat, corn, rice bran, rice bran; rice cake etc. by conventional methods such as milling, soaking, and centrifugation. As a raw material for starch, corn is preferable because it is an inexpensive raw material. By subjecting the cooked starch to a decomposition reaction, the hydrolysis efficiency by amylase is improved.
Yeast density in yeast reaction varies according to the number and activity of amylases which is fixed per yeast 1 cells, it is preferred that the amylase activity in the reaction solution is a density of the order of 0.1~200U / ml, 0.5 More preferably, the density is about ˜100 U / ml.

  Yeast may be included in the reaction solution as it is, or may be immobilized, but is immobilized in that it can be used continuously by continuous reaction, batch reaction or semi-batch reaction. Is preferred. “Immobilization” means a state in which the yeast is not in a free state, for example, a state in which the yeast is bound to, attached to, or incorporated into the carrier. The immobilization method is not particularly limited, and a known microorganism immobilization method can be employed. Examples of such known immobilization methods include a method of holding yeast on a carrier having pores, a method of enclosing yeast with a lattice or microcapsule, and the like.

  As the carrier, a carrier made of a substance insoluble in water or a solvent in the reaction solution is used. Specifically, for example, a porous body made of a resin such as polyvinyl alcohol, polyacrylamide, polyvinyl formal, or cellulose can be suitably used. In addition, foams such as polyurethane foam, polystyrene foam, and silicon foam can be used as the porous body. A porous carrier is desirable because yeast can be successfully grown in the carrier, and further, yeast with reduced activity or dead yeast can be retained for a long time without detachment.

  Although the diameter of the pores (openings) of the porous body varies depending on the type of yeast, it is usually preferably about 50 to 1000 μm, more preferably about 50 to 100 μm. If it is the range of the said opening part diameter, invasion and multiplication of yeast will be easy, and it can hold | maintain yeast with high density.

  The outer shape of the carrier is not particularly limited, and may be any shape such as a spherical shape, a cubic shape or an indefinite shape; a mesh shape or a sheet shape. It is preferable that it is granular in terms of easy preparation. The size of the particles may be, for example, about 2 to 50 mm in diameter in the case of a sphere, and about 2 to 50 mm in the case of a cube.

As a method of immobilizing yeast by inclusion, a method of including yeast in a lattice form using polyacrylamide, calcium alginate, κ-carrageenan, synthetic prepolymer (photocrosslinkable resin prepolymer, urethane prepolymer, etc.); The method of covering this with the microcapsule which consists of a polymer semipermeable membrane by methods, such as a separation method, an interfacial polymerization method, and an underwater drying method, is mentioned. Moreover, the method of bridge | crosslinking yeast mutually is also mentioned.
The decomposition reaction of the reactive starch can be performed in a state where free yeast or immobilized yeast is suspended in the reaction solution in the reaction vessel. Moreover, it can also carry out using the bioreactor which filled the yeast free or fix | immobilized in reaction containers like a column. The reaction may be carried out by any of continuous, batch (batch) and semi-batch methods.

  The reaction solution may contain only starch as a substrate, or in addition to this, may contain a buffer for pH adjustment and a known substance necessary for the survival of the yeast.

  The concentration of the substrate in the reaction solution is usually preferably about 5 to 80% by weight, more preferably about 10 to 30% by weight. If the substrate concentration is too low, the glucose production efficiency is poor. On the other hand, if the substrate concentration is too high, the viscosity of the reaction solution increases, stirring and temperature control become difficult, the osmotic pressure of the reaction solution increases, and a great deal of stress is applied to the yeast. It may become a state to be. Extreme stress triggers deterioration of cell growth or cell death (destruction), and as a result, ideal reaction efficiency cannot be obtained. However, such a problem does not occur within the above range.

  The pH of the reaction solution may be determined in consideration of the optimum pH of the amylase to be used, but is preferably about pH 4 to 8. Since the pH of the reaction solution decreases with the progress of the reaction, it is desirable to adjust the pH range.

  The reaction temperature may be any temperature at which the amylase retained by the yeast is active, and is usually preferably about 20 to 60 ° C. When a thermostable enzyme is used, it can be performed at a higher temperature. In this case, even if the yeast cannot grow, the amylase on the surface layer is fixed while having activity, so that there is no problem in the decomposition reaction.

By monitoring the concentration of each component in the reaction solution over time using, for example, gas chromatography or HPLC, the additional amount of starch, the reaction time, the added amount of pH adjuster, etc. may be determined.
Recovery of glucose Since glucose is produced in the reaction solution by the above reaction, glucose can be recovered from the reaction solution after completion of the reaction by ultrafiltration and concentration. The obtained glucose can be used as it is as a substrate for ethanol fermentation.
(IV) Method for Producing First Xylan Decomposition Product The first method for producing a xylan decomposition product of the present invention comprises a step of decomposing xylan in the presence of the yeast of the present invention and xylosidase or further xylanase, Recovering the decomposition product. As yeast, one in which a polynucleotide encoding arabinofuranosidase is incorporated as a target protein into chromosomal DNA is used.
Xylan such as cedar, beech, bamboo-like wood; weed; rice husk, bran seed husk; rice straw; corn, sugarcane pomace (bagasse), plant meal; soybean meal From plant biomass, hemicellulose containing xylan and arabinoxylan as the main component can be extracted by methods such as explosion, solvent extraction, steam distillation, super-critical carbon dioxide gas extraction, and pressurized hot water extraction.

In the method of the present invention, xylan can be subjected to decomposition in the form of hemicellulose as it is, or xylan separated from hemicellulose can also be subjected to decomposition.
The yeast used is the yeast of the present invention described above. The density of yeast in the reaction solution varies depending on the arabinofuranosidase activity fixed per yeast cell, but a density at which the arabinofuranosidase activity in the reaction solution is about 0.001 to 10 U / ml is preferable. A density of about 5 U / ml is more preferred. Other conditions relating to yeast are as described for the method for producing starch.
Xylanase / xylosidase In order to decompose the β-1,4 bond between xylose in the main chain of xylan, xylosidase or further xylanase is added to the reaction solution. Both xylanase and xylosidase have the activity of cleaving the β-1,4 bond of the xylan main chain. Among them, xylanase is an endo-type enzyme and has an activity of cleaving regardless of the site of the xylan main chain. On the other hand, xylosidase is an exo-type enzyme that cleaves xylose one by one from the end of the xylan main chain. Therefore, xylanase alone cannot produce xylose from xylan. In addition, xylosidase is slow in reaction rate due to the property of decomposing from the xylan end. In the method of the present invention, it is preferable to use a combination of xylanase and xylosidase.

  These enzymes may be derived from any organism. For example, the thing of origin, such as bacteria and mold, can be used. Among them, it is preferable to use a mold (Aspergillus genus) from the viewpoint of easy preparation of a large amount of enzyme.

The amount of xylanase used and the amount of xylosidase used may be such that the activity in the reaction solution is usually about 0.001 to 200 U / ml, preferably about 0.01 to 200 U / ml. These enzyme activities are values measured by the method described in the examples.
The decomposition reaction of xylan by the reaction arabinofuranosidase and xylosidase or further xylanase can be performed in a state where free yeast or immobilized yeast is suspended in the reaction solution in the reaction vessel. Moreover, it can also carry out using the bioreactor which filled the yeast free or fix | immobilized in reaction containers like a column. The reaction may be carried out by any of continuous, batch (batch) and semi-batch methods.

  In the reaction solution, only the substrate may be contained, or in addition to this, a buffer for pH adjustment and a known substance necessary for the survival of the yeast may be contained.

  The concentration of the substrate (xylan) in the reaction solution is usually preferably about 1 to 50% by weight, and more preferably about 1 to 30% by weight. If the substrate concentration is too low, practical xylose production efficiency cannot be obtained. On the other hand, if the substrate concentration is too high, the viscosity of the reaction solution will increase, making stirring and temperature control difficult, and the osmotic pressure of the reaction solution may increase, resulting in a state where a great deal of stress is applied to the yeast. . Extreme stress triggers deterioration of cell proliferation and cell death (destruction), and as a result, ideal reaction efficiency cannot be obtained. However, such a problem does not occur within the above range.

  The pH of the reaction solution is preferably about 4 to 8 including the optimum pH of arabinofuranosidase and xylosidase, or further xylanase. Since the pH of the reaction solution decreases with the progress of the reaction, it is desirable to adjust the pH range.

  The reaction temperature should just be the temperature which the arabinofuranosidase and xylosidase which yeast hold | maintain, and also xylanase show activity, Usually, about 20-60 degreeC is preferable. When a thermostable enzyme is used, it can be performed at a higher temperature. In this case, even if the yeast cannot grow, since the arabinofuranosidase on the surface layer is immobilized with activity, there is no problem in the degradation reaction.

  When cellulose or the like is mixed in addition to xylan in the reaction solution, various sugar hydrolases such as cellulase may be used in combination.

By monitoring the concentration of each component in the reaction solution by measuring with time using, for example, a gas chromatograph or HPLC, the additional amount of the substrate, the reaction time, the added amount of the pH adjuster, etc. may be determined.
Recovery of Xylan Decomposition Products Depending on the source species of xylan, xylose, arabinose, etc. are mainly produced in the reaction solution by the above reaction. Monosaccharides may be separated from the reaction solution after completion of the reaction by centrifugation or the like. Thereby, a xylan decomposition product can be collected. These monosaccharides can be used as they are as substrates for ethanol fermentation.
(V) Method for producing second xylan degradation product The second method for producing xylan degradation product of the present invention is obtained by steps (a) and (a) of decomposing xylan in the presence of the yeast of the present invention. And a step (b) of decomposing the obtained xylan partial decomposition product in the presence of xylosidase or further xylanase, and a step of recovering the xylan decomposition product.
Decomposition process with arabinofuranosidase (process (a))
About a substrate, yeast, and the reaction method, it is the same as that of the manufacturing method of the 1st xylan decomposition product. The arabinofuranosidase activity in the reaction solution is the same as in the first method. The reaction solution after completion of the reaction is subjected to the next step as it is or after recovering and removing arabinose by centrifugation or the like.
Degradation step with xylosidase or further xylanase (step (b))
After decomposing arabinose on the side chain of xylan using the yeast of the present invention, the main chain xylose is decomposed with xylosidase or further xylanase. The reaction method and the recovery method of the xylan decomposition product are the same as in the first method. The enzyme activity in the reaction solution is the same as in the first method.
(VI) Monosaccharide production method The yeast of the present invention can be suitably used for the degradation of other polysaccharides in addition to the degradation of xylan and starch. That is, the method for producing a monosaccharide of the present invention comprises a step of degrading a plant-derived biomass derived polysaccharide in the presence of the yeast of the present invention whose target protein is a sugar hydrolase, and a step of recovering the produced monosaccharide A method including:

  When using practical yeast as yeast, since it has high ethanol tolerance, a reaction liquid can be converted into ethanol as it is using the fermentation ability of this practical yeast.

  Examples of the polysaccharide include cellulose and lignin, which are the main components of woody biomass, in addition to the above-mentioned starch and xylan (the main component of hemicellulose).

  For example, wood such as cedar, beech and bamboo; weeds; seed husks such as rice husks and bran; rice straw; plant meals such as corn, sugarcane pomace (bagasse); cereals such as soybean meal Cellulose and lignin can be extracted from the biomass by methods such as explosion, solvent extraction, steam distillation, supercritical carbon dioxide extraction, and pressurized hot water extraction.

In this case, a laccase gene (Lac) that degrades lignin, a lignin peroxidase gene (LiP), a manganese peroxidase gene (MnP); a cellulase that degrades cellulose, etc. may be used as the polynucleotide encoding the target protein. Degradation conditions can be easily determined by those skilled in the art according to the above-described method for producing a xylan degradation product or glucose.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Production of DNA having a highly active promoter, a secretory signal sequence, a partial sequence of α-agglutinin and a GPI anchor attachment signal sequence in this order)
The procedure for preparing the subject DNA will be described with reference to FIG.
(A) A highly active promoter (SED1 promoter) was obtained according to a conventional method. Briefly, chromosomal DNA of yeast Saccharomyces cerevisiae X21801A was used as a template using two primers 5′-cccgggGAAAAACGACAACATTCCAC-3 ′ (SEQ ID NO: 1) and 5′-gaattcCTTAATAGAGCGAACGTATT-3 ′ (SEQ ID NO: 2). PCR was performed. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then digested with restriction enzymes SmaI and EcoRI to obtain a SmaI-EcoRI fragment. This SmaI-EcoRI fragment contains the promoter region of the SED1 gene. The sequence is shown in SEQ ID NO: 7 in the sequence listing.
(B) A secretory signal sequence of Aspergillus oryzae glucoamylase was obtained according to a conventional method. Briefly, 5′-GAATTCATGCGGAACAACCTTCTTTTT-3 ′ (SEQ ID NO: 3) 5′-CTCGAGGTTGAGATCCGACTGCCTCTT-3 ′ (SEQ ID NO: 4) was synthesized, and using these as a primer, plasmid pNGB1 PCR was performed using (Gene 207 127-134 (1998)) as a template. As PCR conditions, a cycle of 94 ° C./1 minute-52 ° C./1 minute-72 ° C./30 seconds was repeated 30 times. Here, in the design of both primers, recognition sites for restriction enzymes EcoRI and XhoI were added in order to fuse the secretory signal sequence to the target protein later.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit), and then cleaved with restriction enzymes EcoRI and XhoI to obtain an about 100 bp glucoamylase secretion signal sequence. The sequence is shown in SEQ ID NO: 8 in the sequence listing.
(C) In order to obtain a sequence having a gene (320 amino acids) having a sequence encoding a part of the C-terminus of α-agglutinin and a GPI anchor attachment signal sequence, chromosomal DNA was isolated from yeast Saccharomyces cerevisiae X21801A by a conventional method. Separately, PCR was performed using two primers, 5′-ctcgagAGCGCCAAAAGCTCTTTTATC-3 ′ (SEQ ID NO: 5) and 5′-ggtaccTTTGATTATGTTCTTTCTAT-3 ′ (SEQ ID NO: 6). As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times.

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

  The target DNA consists of the highly active promoter obtained in (A), the secretory signal sequence obtained in (B), and the XhoI-KpnI fragment obtained in (C) in a conventional manner using a DNA ligation enzyme (T4 Ligase). It can be obtained by connecting using, for example.

  The obtained DNA of interest was inserted into the SmaI and KpnI cleavage sites of plasmid pRS406 to obtain the desired plasmid pRS406 Psed1-CAS1.

(Comparison of cell surface display efficiency by fluorescent protein)
According to a conventional method, a fluorescent protein gene (EGFP) was obtained. Briefly, 5'-ctcgagATGGTGAGCAAGGGCGAGG-3 '(SEQ ID NO: 10) 5'-ctcgagCTTGTACAGCTCGTCCATG-3' (SEQ ID NO: 11) was synthesized and used as a primer, plasmid pEGFP (sold from CLONTECH) as a template PCR was performed. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./1 min was repeated 30 times. Here, in designing both primers, a restriction enzyme XhoI recognition site was added and a translation stop codon was removed in order to fuse a fluorescent protein with a sequence encoding a secretion signal and a part of α-agglutinin C-terminal. .

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain a fluorescent protein sequence of about 0.7 Kbp. The sequence is shown in SEQ ID NO: 12 in the sequence listing.

  The fluorescent protein sequence was inserted into the XhoI cleavage site of plasmid pRS406 Psed1-CAS1 to obtain the desired plasmid pRS406 Psed1-EGFP-CAS1.

Plasmid pRS406 Psed1-EGFP-CAS1 was isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into yeast Saccharomyces cerevisiae GRI-117-U by the lithium acetate method. Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, plasmid pRS406 Psed1-EGFP-CAS1 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. The strain thus obtained was named Saccharomyces cerevisiae GRI-117-U (EGFP) (hereinafter simply referred to as GRI-117-U (EGFP)).
<Measurement of growth rate>
GRI-117-U (EGFP) and a strain obtained by transforming the laboratory yeast Saccharomyces cerevisiae MT8-1 prepared for comparison between the present invention and the prior art with pRS406 Psed1-EGFP-CAS1 (hereinafter simply referred to as MT8- 1 (EGFP)) is suspended in SD liquid medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, respectively, and shaken at 30 ° C. The growth rates of both strains were compared by culturing and measuring the absorbance at 600 nm over time.

The results are shown in FIG. GRI-117-U (EGFP), which is a practical yeast, showed about twice the cell growth as compared to MT8-1 (EGFP), which is a laboratory yeast.
<Measurement of fluorescence intensity>
GRI-117-U (EGFP), Saccharomyces cerevisiae GRI-117-U as a control, and MT8-1 (EGFP) were 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), cultured in an SD liquid medium containing 2% glucose, and centrifuged to separate the medium and cells.

  After suspending the cells of GRI-117-U (EGFP) and MT8-1 (EGFP) in an appropriate amount of water, their fluorescence intensity is measured using a commercially available fluorescent multiplate reader (TECAN Multi Plate Reader Optima Fluor etc. ).

  The results are shown in FIG. The fluorescence intensity shown in FIG. 5 is a value obtained by subtracting the fluorescence intensity of the parent strain (GRI-117-U or MT8-1) from the fluorescence intensity of each strain as a control. As a result, GRI-117-U (EGFP) had a fluorescence signal about 10% stronger than MT8-1 (EGFP).

(Production of yeast having glucoamylase on the cell surface)
According to a conventional method, Aspergillus oryzae glucoamylase was obtained. Briefly, 5′-ctcgagCAGGCGACTGGTCTAGATACC-3 ′ (SEQ ID NO: 13) 5′-ctcgagCCGCCAAACATCGCTCTGCAC-3 ′ (SEQ ID NO: 14) was synthesized, and this was used as a primer for plasmid pYGA1 PCR was performed using (Agric. Biol. Chem. 55 941-949 (1991)) as a template. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times. Here, in designing both primers, a recognition site for the restriction enzyme XhoI was added to eliminate the translation termination codon in order to later fuse glucoamylase with a secretion signal and a sequence encoding part of the C-terminus of α-agglutinin. did.

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain an about 1.8 Kbp glucoamylase sequence. The sequence is shown in SEQ ID NO: 15 in the sequence listing.

  Glucoamylase DNA was inserted into the XhoI cleavage site of plasmid pRS406 Psed1-CAS1 to obtain the desired plasmid pRS406 Psed1-glaA-CAS1.

  Plasmid pRS406 Psed1-glaA-CAS1 was isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into yeast Saccharomyces cerevisiae GRI-117-U by the lithium acetate method. Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, plasmid pRS406 Psed1-glaA-CAS1 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. The strain thus obtained was named Saccharomyces cerevisiae GRI-117-U (glaA) (hereinafter simply referred to as GRI-117-U (glaA)).

  GRI-117-U (glaA) is cultured in an SD liquid medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 2% glucose, and centrifuged. It isolate | separated into the culture medium and the microbial cell, and measured each glucoamylase activity. As a control, Saccharomyces cerevisiae GRI-117-U was used. As a result, the control showed no glucoamylase activity in the medium and cells. Almost no glucoamylase activity was found in the transformed yeast GRI-117-U (glaA) medium, but the yeast GRI-117-U (glaA) cells had glucoamylase activity.

Further, the glucoamylase activity of GRI-117-U (glaA) cells was about 5 × 10 ⁻³ U per 1 mg cells.

The glucoamylase activity was measured by the following method.
<Measurement of glucoamylase activity>
For measurement of glucoamylase activity, Kikkoman “Saccharification power measurement kit” is used. Briefly, after adding an appropriate amount of bacterial cells or culture supernatant to an acetic acid buffer containing 4-Nitrophenyl β-L-maltoside, leave it at 37 ° C for 10 minutes and determine the amount of 4-Nitrophenol released. Calculated from the absorbance at 415 nm. Glucoamylase activity was defined as the amount of enzyme required to produce 1 μmol of 4-Nitrophenol per minute from 1U = 4-Nitrophenyl β-L-maltoside.

(Immobilization of yeast with glucoamylase on the cell surface)
0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 6mm square polyurethane foam BSPs in a bubble column type culture tank containing 3L of SD medium containing 2% glucose, The cells were added at 1,000 cells / L and cultured with yeast GRI-117-U (glaA) at an aeration rate of 6 to 20 L / min. Thereby, yeast GRI-117-U (glaA) was fixed to the polyurethane foam.

  The glucoamylase activity of this yeast was 1 U per polyurethane foam.

(Degradation of starch by yeast having glucoamylase on immobilized cell surface)
To 3,000 yeast GRI-117-U (glaA) fixed to the polyurethane foam obtained in Example 4, 50 g of corn starch and 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077 1 L of medium containing% CSM-URA (BIO101) was added and contacted at 30 ° C. for 24 hours in a bubble column type culture tank with an aeration rate of 3 to 20 L / min. During the reaction, the pH was controlled at 5.0 to 6.0 using KOH. After collecting the reaction solution of the first cycle, a new reaction solution was added to perform a reaction of 2 cycles, and this was performed up to 5 cycles.
<Measurement of ethanol production>
As a result of analyzing the composition of the reaction solution after completion of the first cycle when yeast GRI-117-U (glaA) was used, it was found that 17 g of ethanol was produced in the culture tank. The ethanol concentration in the reaction solution after completion of the first cycle was about 1.7% by volume.

  The immobilized yeast GRI-117-U (glaA) expressed glucoamylase activity without any decrease in activity after the second cycle. When the yeast released from the immobilized cells was collected and the activity was measured, the glucoamylase activity was greatly reduced.

  For comparison between the present invention and the prior art, a strain obtained by transforming the laboratory yeast Saccharomyces cerevisiae MT8-1 with pRS406 Psed1-glaA-CAS1 was also prepared (hereinafter simply referred to as MT8-1 (glaA)). The same operation was performed. As a result of analyzing the composition of the reaction solution after completion of the first cycle, the ethanol concentration in the reaction solution was about 0.6% by volume.

  The result is shown in FIG. GRI-117-U (glaA) produced about three times as much ethanol as MT8-1 (glaA).

(Production of yeast having arabinofuranosidase on the cell surface)
According to a conventional method, cDNA of Aspergillus oryzae arabinofuranosidase was obtained. Briefly, first, total RNA was extracted from Aspergillus oryzae, and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. More specifically, 5′-ctcgagATGACTACCTTCACGAAAC-3 ′ (SEQ ID NO: 16) 5′-ctcgagAGCCTTCCATCTCAGGAGA-3 ′ (SEQ ID NO: 17) was synthesized, and PCR was carried out using this cDNA mixture as a template. As PCR conditions, a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 2 minutes was repeated 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes. Here, in designing both primers, the restriction enzyme site XhoI was added and the translation termination codon was removed in order to later fuse arabinofuranosidase with a part of the C-terminus of α-agglutinin.

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain an about 1.5 kbp arabinofuranosidase cDNA fragment. The sequence is shown in SEQ ID NO: 18 of the sequence listing.

  The arabinofuranosidase DNA was inserted into the XhoI cleavage site of plasmid pRS406 Psed1-CAS1 to obtain the desired plasmid pRS406 Psed1-abfA-CAS1.

Plasmid pRS406 Psed1-abfA-CAS1 was isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into yeast Saccharomyces cerevisiae GRI-117-U by the lithium acetate method. Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, the plasmid pRS406 Psed1-abbA-CAS1 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named Saccharomyces cerevisiae GRI-117-U (abfA) (hereinafter simply referred to as GRI-117-U (abfA)). The obtained yeast GRI-117-U (abfA) was cultured in an SD liquid medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 2% glucose, Centrifugation was performed to separate the medium and the cells, and each arabinofuranosidase activity was measured. As a control, Saccharomyces cerevisiae GRI-117-U was used. As a result, the control showed no arabinofuranosidase activity in the medium and cells. Almost no arabinofuranosidase activity was observed in the medium of transformed yeast GRI-117-U (abfA), but yeast GRI-117-U (abfA) had arabinofuranosidase activity. Was.
<Detection of arabinofuranosidase activity>
The presence or absence of arabinofuranosidase activity was determined by adding the cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl α-L-arabinofuranoside and allowing to stand at 50 ° C. for 30 minutes. The released 4-Nitrophenol was visually confirmed. Since free 4-Nitrophenol exhibits a yellow color, this color is confirmed.

(Immobilization of yeast having arabinofuranosidase on the cell surface)
0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 6mm square polyurethane foam BSPs in a bubble column type culture tank containing 3L of SD medium containing 2% glucose, The cells were added at 1,000 cells / L and cultured with the yeast GRI-117-U (abfA) at an aeration rate of 6 to 20 L / min. Thereby, yeast GRI-117-U (abfA) was fixed to the polyurethane foam.

  The arabinofuranosidase activity of this yeast was 0.01 U per polyurethane foam.

(Degradation of xylan by yeast with arabinofuranosidase on immobilized cell surface)
To 3,000 yeast GRI-117-U (abfA) fixed to the polyurethane foam obtained in Example 6, reaction solution: crude xylan 100 g, xylanase (total activity 150 U amount), xylosidase (total activity 150 U amount), And 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101), 3 L of SD medium containing 2% glucose, and bubble column culture at an aeration rate of 3-20 L / min It was made to contact at 30 degreeC for 24 hours within a tank. During the reaction, the pH was controlled at 5.0 to 6.0 using KOH.

  After collecting the reaction solution of the first cycle, a new reaction solution was added to perform a reaction of 2 cycles, and this was performed up to 5 cycles. As a result of analyzing the composition of the reaction solution after completion of the first cycle, it was found that 10 g of xylose was produced in the culture tank.

The immobilized yeast GRI-117-U (abfA) expressed arabinofuranosidase activity without any decrease in activity after the second cycle.
<Quantification of arabinofuranosidase activity in bacterial cells>
The arabinofuranosidase activity is determined by adding the bacterial cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl α-L-arabinofuranoside, leaving it at 50 ° C. for 30 minutes, and releasing it. The amount of 4-Nitrophenol was determined. The arabinofuranosidase activity was defined as 1U = the amount of enzyme required to produce 1 μmol of 4-Nitrophenol per minute from 4-Nitrophenyl α-L-arabinofuranoside.
<Quantification of xylanase activity>
Xylanase activity was measured by adding the bacterial cells, culture supernatant or purified enzyme solution to 50 mM acetate buffer (pH 6.5) containing 1% birchwood glucuronoxylan (Sigma), and allowing to stand at 37 ° C. for 10 minutes. The reducing power of the released sugar was quantified by the Somogy Nelson method. Xylanase activity was defined as 1U = the amount of enzyme that produces a reducing power equivalent to 1 μmol of xylose per minute from bircwood glucuronoxylan.
<Quantification of xylosidase activity>
Xylosidase activity was determined by adding the bacterial cell or culture supernatant purified enzyme solution to 50 mM citrate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl β-D-xyloside, and allowing to stand at 50 ° C. for 5 minutes. It was determined from the amount of released 4-Nitrophenol. Xylosidase activity 1U = the amount of enzyme required to produce 1 μmol of 4-Nitrophenol per minute from 4-Nitrophenyl β-D-xyloside.

It is a figure which shows the structure of the expression unit in the expression vector of this invention. It is a figure explaining the mode of processing and intracellular transport of GPI anchoring protein. It is the figure which showed the preparation procedure of DNA which has a highly active promoter, a secretion signal sequence, a partial sequence of α-agglutinin, and a GPI anchor attachment signal sequence in this order. It is the figure which compared the growth rate of GRI-117-U (EGFP) with the growth rate of strain MT8-1 (EGFP) which transformed laboratory yeast Saccharomyces cerevisiae MT8-1 with pRS406 Psed1-EGFP-CAS1. It is the figure which compared the fluorescence intensity of GRI-117-U (EGFP) with the fluorescence intensity of MT8-1 (EGFP). It is the figure which compared the ethanol production amount from the corn starch of GRI-117-U (glaA), and the ethanol production amount from the corn starch of MT8-1 (glaA).

Claims (10)

In order from the 5 ′ end, a highly active promoter selected from the group consisting of the yeast SED1 promoter, GAPDH promoter, PGK1 promoter, and ADH1 promoter, a secretory signal sequence that functions in yeast cells, a sequence encoding the target protein, and a cell surface localized protein A polynucleotide comprising a sequence encoding a portion of the GPI anchor attachment signal sequence. The polynucleotide according to claim 1, wherein the highly active promoter is a yeast SED1 promoter. A chromosomally integrated expression vector comprising the polynucleotide according to claim 1 or 2. A yeast in which the polynucleotide of claim 1 or 2 or the expression vector of claim 3 is incorporated into chromosomal DNA. The yeast according to claim 4, which is a practical yeast. The target protein is a sugar hydrolase, The production of a monosaccharide comprising a step of degrading a polysaccharide derived from plant biomass and a step of recovering the produced monosaccharide in the presence of the yeast according to claim 4 or 5 Method. The method for producing glucose comprising a step of degrading starch in the presence of yeast according to claim 4 or 5, and a step of recovering the produced glucose. The method according to claim 7, wherein the amylase is glucoamylase. The yeast according to claim 4 or 5, wherein the target protein is arabinofuranosidase, and xylan decomposition comprising the step of decomposing xylan in the presence of xylosidase or further xylanase, and recovering the produced xylan degradation product Manufacturing method. The target protein is arabinofuranosidase, the step (a) for degrading xylan in the presence of yeast according to claim 4 or 5, and the xylan partial degradation product obtained by step (a) is converted into xylosidase or further xylanase A method for producing a xylan decomposition product, comprising the step (b) of decomposing in the presence of water and the step of recovering the generated xylan decomposition product.

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