JP2011019429A - Yeast holding cellulase on the cell surface and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a cellulase suitable for being used together with other cellulases, when held on the cell surface of the yeast, and utilize the cellulase. <P>SOLUTION: There is provided a yeast holding, on the cell surface of the yeast, one or more cellulases selected from the group consisting of cellobiohydrolase Cel 5F and endoglucanase Cel 5C belonging to glucosidase family-5, endoglucanase Cel 8A belonging to glucosidase family-8, endoglucanase Cel 9D, endoglucanase Cel 9R, cellobiohydrolase Cel 9K, and cellobiohydrolase Cbh A belonging to glucosidase family-9, and a combination thereof. The yeast or the combination thereof is used. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to yeast that retains cellulase on the cell surface and use thereof.

  In recent years, recyclable biomass resources have attracted attention in consideration of future depletion of petroleum resources. Cellulose, which is a basic component of plant biomass, is decomposed by a cooperative or stepwise reaction by a plurality of types of cellulases. For efficient use of cellulose, selection of a combination of cellulases used for cellulose degradation is also considered important.

  On the other hand, a technique for presenting a protein such as an enzyme on the surface of a cell such as yeast is known. Attempts have also been made to present cellulase on the surface layer of yeast having high fermentability to decompose and saccharify cellulose derived from biomass or the like (Non-patent Document 1). In this document, a combination of several enzymes selected from β-glucosidase I, endoglucanase II (GH family 5) and cellobiohydrolase II (GH family 6) derived from Trichoderma reesei was surface-displayed. It is described. Moreover, it is described that the yeast which surface-displayed both endoglucanase and cellobiohydrolase decomposed amorphous cellulose better than the yeast which displayed only endoglucanase.

  In addition, an attempt has been made to construct an artificial protein complex of a scaffolding protein component and a cellulase in a cellulase complex called cellulosome in the cell surface layer such as Clostridium bacteria (Non-patent Document 2). , 3). In these documents, artificial scaffolding proteins and artificial chimeric cellulases produced by genetic engineering in Escherichia coli and Bacillus subtilis are combined in vitro to construct artificial protein complexes. Is described. Further, it is described that cellulose was decomposed with such an artificial protein complex to evaluate the effect of the combination.

Fujita, Y., et al., App 70 (2) 1207-1212 (2004) Fierobe, H. P., et al., J. Biol. Chem., 277 (51), 49621-630 (2002) Fierobe, H. P., et al., J. Biol. Chem., 280 (16), 16325-334 (2005)

  However, in Non-Patent Document 1, it is a well-known fact that, according to endoglucanase and cellobiohydrolase, it is retained in the cell surface layer of yeast that cellulose can be decomposed more effectively than endoglucanase alone. It is only shown in the state. Non-Patent Documents 2 and 3 are merely evaluations of protein complexes produced in vitro by producing the complex in E. coli. Therefore, it is not possible to predict that a preferable combination of cellulases in the evaluation is a preferable combination of cellulases when retained on the surface of yeast. This is because there is a sugar chain modification of a protein during expression in yeast or surface display, and the enzyme activity decreases with a low probability. Furthermore, Non-Patent Documents 2 and 3 are evaluation results for cellulases derived from some cellulases, that is, Clostridium cellulolyticum, and are immediately applied to cellulases derived from other microorganisms and other cellulases. It can't be done.

  Therefore, the present invention aims to find a cellulase suitable for use in combination with another cellulase when it is held on the cell surface of yeast, and to use it.

  The present inventors have searched for various cellulases suitable for expression in yeast and retention on the surface layer, and have found a plurality of enzymes that have a synergistic effect on cellulose degradation by combining with other cellulases. The present inventors have completed the present invention based on these findings.

  According to the present invention, cellobiohydrolase Cel 5F belonging to saccharolytic enzyme family 5, endoglucanase Cel 5C, endoglucanase Cel 8A belonging to saccharolytic enzyme family 8, endoglucanase Cel belonging to saccharolytic enzyme family 9 One or more cellulases selected from the group consisting of 9D, the same endoglucanase Cel 9R, the same cellobiohydrolase Cel 9K and the same cellobiohydrolase Cbh A (hereinafter also referred to as cellulase group) are retained on the cell surface layer. Yeast or a combination thereof is provided.

  In the yeast of the present invention, the cellulase retained in the cell surface layer of the yeast is preferably at least two kinds including at least a processive cellulase. Moreover, it is preferable that the cellulases retained in the cell surface layer of the yeast are at least two kinds including cellulases belonging to the saccharide-degrading enzyme family 9. Furthermore, the cellulase retained in the cell surface layer of the yeast is one or two selected from the cellulase shown in the first column of Table 1 below and the cellulase described in the second column corresponding to the first column. Preferably, the cellulase shown in the first column of Table 2 below and one or more selected from the cellulases described in the second column corresponding to the first column are included. Is also preferable. Note that Cel 9D in this specification is also expressed as Cel 9A in CAZy's GHF server (http://www.cazy.org/fam/acc_GH.html) described later. Further, Cbh A in this specification is also expressed as Cbh 9A in CAZy.

In the yeast of this invention, it is preferable that the said cellulase hold | maintained at the cell surface layer of the said yeast has the following relationship with the base sequence or amino acid sequence represented by the sequence number of the following Table 3, respectively.
(A) Cellulase encoded by a gene consisting of the base sequence represented by each sequence number described in Table 3 (b) DNA complementary to the DNA comprising the base sequence represented by each sequence number described in Table 3 A cellulase encoded by a gene that hybridizes under stringent conditions with cellulase activity (c) a base sequence having 70% or more homology to the base sequence represented by each sequence number in Table 3 A cellulase having a corresponding cellulase activity encoded by a gene comprising: (d) a cellulase having an amino acid sequence represented by each SEQ ID NO: listed in Table 3 (e) an amino acid sequence represented by each SEQ ID NO: listed in Table 3 In which one or more amino acids are deleted, substituted or added and have a corresponding cellulase activity (F) cellulase having corresponding cellulase activity has an amino acid sequence having 70% or more identity to the amino acid sequence represented by the SEQ ID NO set forth in Table 3

  The yeast of the present invention or a combination thereof The cellulase retained on the cell surface of the yeast is preferably derived from Clostridium, more preferably from Clostridium thermocellum. The cellulase retained on the cell surface of the yeast is also preferably retained on the cell surface via a protein derived from a scaholdin protein of a cellulosome-producing microorganism, more preferably Clostridium thermocellum. Derived from the Scaffoldin protein. Further, the protein derived from the scaffolding protein is preferably derived from a type I scaffolding protein.

  Furthermore, it is preferable that the yeast of the present invention or a combination thereof includes a yeast that secrete-produces β-glucosidase.

  According to the present invention, there is provided a method for producing a cellulose degradation product from cellulose using cellulase, wherein the yeast of the present invention or a combination thereof is used, and the two or more cellulases retained by the yeast or the combination thereof are used. And a step of decomposing cellulose.

  According to the present invention, there is provided a method for producing a useful substance from cellulose, wherein the yeast of the present invention or a combination thereof is used to decompose cellulose using two or more cellulases held by the yeast or the combination thereof. And a step of producing a useful substance by the yeast or other microorganisms using a carbon source containing a cellulose degradation product obtained in this manner.

It is a figure which shows the structure of 2 micrometers vector used in an Example. It is a figure which shows the activity evaluation result based on the CMC decomposition | disassembly activity of various cellulases hold | maintained on the surface layer of yeast. It is a figure which shows the activity evaluation result based on 0.5% PSC decomposition | disassembly activity when the yeast produced in Example 2 was individual and it combined 2 types. It is a figure which shows the activity evaluation result based on 1% Avicel decomposition | disassembly activity when the yeast produced in Example 2 was individual and it combined 2 types. It is a graph which shows the decomposition | disassembly activity of crystalline cellulose (trade name Avicel) when using the yeast produced in Example 5 (simultaneous expression of two types of cellulases). It is a graph which shows the ethanol density | concentration in a culture medium when it ferments with the culture medium containing crystalline cellulose using the co-expression yeast which is produced in Example 5 and has the highest cellulose decomposition activity.

  The present invention relates to a yeast or a combination thereof that retains one or more cellulases selected from the cellulase group in a cell surface layer. The yeast of the present invention or a combination thereof retains a cellulase that is preferable for expressing the activity retained in the cell surface layer of the yeast, and can efficiently decompose cellulose. In particular, among these cellulases, a high cellulolytic activity can be obtained by using a processive cellulase and / or a cellulase belonging to the saccharide-degrading enzyme family 9 in combination with other cellulases.

  When cellulose is decomposed by cellulase held on the surface layer of yeast or the like, the combination of the cellulases is extremely important. The expression of many kinds of enzymes may reduce the fermentation ability and growth ability of yeast, and the use of too many kinds of enzymes is not preferable in terms of cost.

  Hereinafter, the yeast of the present invention or a combination thereof and use thereof will be described in detail. In the present specification, the yeast combination of the present invention means a combination (mixture or set) of two or more yeasts having different types of cellulase to be held.

(Cellulase)
Cellulases retained by the yeast of the present invention on the cell surface include cellobiohydrolase Cel 5F belonging to saccharide-degrading enzyme family 5, endoglucanase Cel 5C, endoglucanase Cel 8A belonging to saccharide-degrading enzyme family 8, and saccharide-degrading enzyme It is selected from the group consisting of endoglucanase Cel 9D belonging to family 9, endoglucanase Cel 9R, cellobiohydrolase Cel 9K and cellobiohydrolase Cbh A.

  These cellulases are classified into glycoproteinase family (GHF) servers (http: //www.cazy), which are protein structural classes classified by analyzing the amino acid sequence of carbohydrate-related enzymes and the three-dimensional structure of the catalytic site. .org / fam / acc_GH.html, Henrissat B (1991) A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 280: 309-316). The cellulase used in the present invention is cellobiohydrolase (Enzyme commission (EC) number based on classification by the International Commission on Biochemical Enzymes (EC) number: EC 3.2.1.91) or endoglucanase (same; EC 3.2.1.4), The GHFs to which the same enzyme name (for example, an enzyme name such as Cel 5F) is assigned or those to which the same enzyme name is assigned in the future are included.

  Cellulase is known to have a mode of performing a hydrolysis reaction while continuously moving while capturing a cellulose molecular chain, and this type of degradation is called processive degradation. From the degradation aspect, the cellulase called cellobiohydrolase (CBH) is considered to be a processive cellulase. Cellulase called endoglucanase is generally considered to be a non-processive cellulase, but partly a processive type (Cel 9R (PEG) in Table 4) is known.

  Cellulases used in the present invention are shown in Table 4 together with GHF, enzyme name, and enzyme type. As a typical example, the base sequence and amino acid sequence of cellulase derived from Clostridium thermocellum disclosed in Examples are also shown. Each of these sequences includes an amino acid sequence corresponding to the signal peptide and a base sequence encoding it. The signal peptide can be predicted by, for example, a known secretory signal sequence prediction program such as SignalP 2.0. In determining the identity of the amino acid sequence and base sequence, which will be described later, a mature peptide excluding the signal peptide predicted by such a program may be used.

  All of these enzymes are suitable for degrading cellulose by being retained on the surface of yeast cells, and among them, processive cellulases and cellulases belonging to GHF9 can be preferably used. These cellulases can exhibit high cellulolytic activity by combining with other processive or non-processive cellulases shown in Table 4 and other cellulases belonging to other GHF9, GHF5, and GHF8.

  As shown in the following table, a processive cellulase is effective for decomposing amorphous cellulose, and Cel 9R (PEG) is excellent in synergistic effect and decomposition activity with non-processive cellulase. Cel 9R exhibits a good synergistic effect and cellulolytic activity with cellulases belonging to GHF5 such as Cel 5F (CBH), cellulases belonging to GHF8 such as Cel 8A (EG) and cellulases belonging to GHF9 such as Cel 9D (EG). can do. Among them, it is preferably combined with Cel 9D and / or Cel 8A.

  As shown in Table 1, Cel 5F (CBH) belonging to GHF5 is suitable for decomposing amorphous cellulose. Cel 5F can exhibit a good synergistic effect and cellulolytic activity together with cellulases belonging to GHF8 such as Cel 8A (EG) and cellulases belonging to GHF9 such as Cel 9D (EG) and Cel 9R (PEG). Among them, it is preferably combined with Cel 9D.

  As shown in Table 2, when synthesizing crystalline cellulose, a high synergistic effect and decomposition activity can be exhibited by combining a processive cellulase belonging to GHF9 with another cellulase. That is, Cel 9R (PEG), Cel 9K (CBH) and Cbh A (CBH) belonging to GHF9 are suitable. Enzymes belonging to GHF9 such as Cel 5F (CBH) and cellulases belonging to GHF8 such as Cel 8A (EG) can exhibit a high synergistic effect and cellulolytic activity. These enzymes can also exhibit high crystalline cellulolytic activity when used in combination with Cel 9D (EG), which is an endoglucanase belonging to each other or other GHF9.

  Cel 9R, Cel 9K and Cbh A belonging to GHF9 all show low degradation activity on crystalline cellulose alone, but Cel 8A which is an endoglucanase belonging to GHF8 and Cel 5F which is an endoglucanase belonging to GHF5. In combination with Cel 9D, which is an endoglucanase belonging to GHF, high crystalline cellulose degrading activity can be exhibited.

  From the above, the following cellulase combinations can be mentioned as suitable combinations. When the enzyme in the left column is used, the enzyme that can be preferably combined to decompose amorphous cellulose is shown in the middle column, and the enzyme that can be preferably combined to decompose crystalline cellulose is shown in the right column. Only one type of enzyme shown in each of the middle column and the right column may be combined, two or more types may be used in combination, or all may be used.

  These enzymes can be used in combination with the enzymes themselves, but can be used by being presented on the surface of a cell such as yeast as described later. Even in this case, cellulases can be combined in various ways. Depending on the cells used, only some enzymes (cellulases) may be produced preferentially. Therefore, when the cells are used, the same type of cellulase as the preferentially produced cellulase is used instead of the same type of cellulase. It is preferable to express in yeast. For example, according to the knowledge of the present inventors, in yeast, endoglucanase such as Cel A tends to be expressed in preference to other cellulases. For this reason, when Cel A and another cellulase are expressed in combination in the same yeast, the Cel A activity is enhanced, and there is a tendency that a synergistic effect with other cellulase is hardly exhibited. Therefore, in such a yeast, when two or more types of cellulases are simultaneously expressed in the same yeast, it is preferable to express cellulases other than Cel A, for example, cellulases belonging to GHF9 in combination. Cel A may be presented on the surface of another yeast cell or added externally.

  The cellulase is preferably derived from Clostridium thermocellum. The present inventor examined cellulase groups obtained from Clostridium cellulolyticum and Clostridium cellulovorans in addition to clostridium thermocellum. Cellulases belonging to GHF5, GHF8, GHF9 and GHF48 Were able to be expressed in yeast, but only Clostridium thermocellum was able to confirm a wide and good cellulase activity for cellulases belonging to GHF5, 8 and 9. However, cellulases derived from other microorganisms exhibited activity only for a small part of cellulases when produced in yeast.

  Examples of the cellulase include proteins each having an amino acid sequence represented by a sequence number shown in Table 4. Moreover, the protein of the various aspects which have a fixed relationship with these amino acid sequences may be sufficient.

  The cellulase used in the present invention is a protein having a certain relationship with the sequence information represented by the sequence numbers shown in Table 4 and having a specific cellulase activity (activity as endoglucanase and cellobiohydrolase). May be. An example of such an embodiment is a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by each SEQ ID No. shown in Table 4.

  The phrase “having cellulase activity” is sufficient if each cellulase has its own cellulase activity. Preferably, it has the same level as or higher than the protein having the amino acid sequence represented by each amino acid sequence shown in Table 4. is there. Whether the cellulase activity is equal to or higher than that is preferably, for example, 70% or more of the cellulase activity of the protein when the protein comprising the amino acid sequence represented by the related SEQ ID NO is expressed in yeast, More preferably, it is 80% or more, More preferably, it is 90% or more, Most preferably, it is 100% or more.

  As for the amino acid mutation relative to the amino acid sequence represented by each SEQ ID NO, that is, deletion, substitution or addition may be any one kind, or two or more kinds may be combined. The total number of these mutations is not particularly limited, but is preferably about 1 or more and 10 or less. More preferably, it is 1 or more and 5 or less.

  As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups. (Glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

  As another embodiment, the cellulase used in the present invention has an amino acid sequence having 70% or more identity to the amino acid sequence represented by each SEQ ID NO shown in Table 4, and has cellulase activity. Examples include proteins. The identity is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and still more preferably 95% or more. Most preferably, it is 98% or more.

  As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. In the art, “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance. Similarity is also the correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of 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 BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  In yet another aspect, a unique cellulase encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by each SEQ ID No. shown in Table 4 Examples include proteins having activity.

  The stringent condition refers to, for example, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, a nucleic acid having a high base sequence identity, that is, a DNA comprising a base sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity with a predetermined base sequence In which the complementary strands of the nucleic acids hybridize and the complementary strands of nucleic acids having lower homology do not hybridize. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Furthermore, under stringent conditions, the filter washing conditions after hybridization are usually such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC. In addition, from the above, as another embodiment, it has a base sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity with a predetermined base sequence. Examples include proteins encoded by DNA and having cellulase activity.

  Such various cellulases can be obtained by obtaining a cellulase gene encoding such cellulase, and can be expressed and retained in yeast. The cellulase gene is obtained by PCR amplification using, for example, DNA extracted from a predetermined yeast, various cDNA libraries or genomic DNA libraries as a template using primers designed based on the sequence of SEQ ID NO: 1 or the like. By performing, it can be obtained as a nucleic acid fragment. Further, it can be obtained as a nucleic acid fragment by performing hybridization using a nucleic acid derived from the library or the like as a template and a DNA fragment which is a part of the cellulase gene as a probe. Alternatively, the cellulase gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  In addition, cellulase genes corresponding to the cellulases of these various modes are obtained by, for example, converting a DNA encoding the amino acid sequence represented by each sequence number shown in Table 4 into a conventional mutagenesis method, site-specific mutagenesis method, error It can be obtained by modifying by a molecular evolution method using prone PCR. As such a technique, a known technique such as the Kunkel method or the Gapped duplex method, or a similar method can be mentioned. For example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant-K (manufactured by TAKARA) ), Mutant-G (manufactured by TAKARA)), or the like, or the LA PCR in vitro Mutagenesis series kit from TAKARA.

  In addition, those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, cellulase genes of various aspects can be obtained based on the respective SEQ ID NOs shown in Table 4.

  In addition, the amino acid sequence represented by each SEQ ID No. shown in Table 4 or the base sequence encoding the amino acid sequence having a certain relationship with the amino acid sequence as described above is the amino acid sequence of the protein according to the degeneracy of the genetic code. Without changing the sequence, at least one base of a base sequence encoding a predetermined amino acid sequence can be replaced with another type of base. Therefore, the cellulase gene used in the present invention also includes a cellulase gene encoding a base sequence converted by substitution based on the degeneracy of the genetic code. The gene may be cDNA or the like in addition to genomic DNA.

  Cellulase can be synthesized by known protein synthesis methods or genetic engineering, and can be expressed in yeast by introducing such cellulase gene into yeast and transforming it.

  Cellulase is retained in the yeast cell surface in various forms. One is a form in which cellulase is retained on the yeast cell surface as it is using a known yeast cell surface display system. Two or more types of cellulases may be retained on the cell surface of the same yeast, or may be retained on the cell surface of different yeasts.

  When cellulase is displayed on the cell surface of yeast, it is preferable that the cellulase further has a cell surface binding domain necessary for cell surface display. As the yeast surface display system, for example, α-agglutinin which is a surface protein or a receptor thereof can be used. For example, in addition to the secretion signal, a peptide comprising 320 amino acid residues on the C-terminal side of α-agglutinin which is an aggregating protein is used. Polypeptides and methods for displaying a desired protein on the cell surface are disclosed in WO01 / 79483, JP2003-235579, WO2002 / 042483, WO2003 / 016525, and JP2006-136223. Publication, Fujita et al. (Fujita et al., 2004. Appl Environ Microbiol 70: 1207-1212 and Fujita et al., 2002. Appl Environ Microbiol 68: 5136-5141.), Murai et al., 1998. Appl Environ Microbiol 64: 4857-4861 Is disclosed. For example, the signal sequence may be an element incorporated into the vector, but may be a part of the cellulase gene. A cell surface display system for proteins using agglutinin can be obtained, for example, as a display kit for yeast containing pYD1 vector and EBY100 Saccharomyces cerevisiae from Invitrogen. As the cell surface display system, a system using cell surface proteins such as SAG1 and FLO1 to FLO11 can be used.

  Another form in which cellulase is retained on the surface of yeast cells is a form retained via a protein derived from the cellulosomal scaffoldin protein. In order to retain such cellulase, yeast having a protein derived from the scaffolding protein on the cell surface layer is used. Scaffoldin proteins include type I and type II. Proteins derived from type I scaffoldin proteins are hereinafter referred to as first skeletal proteins, and proteins derived from type II scaffoldin proteins are referred to below. , Referred to as the second scaffold protein.

  Cellulosome is formed outside the cells by anaerobic bacteria or anaerobic filamentous fungi, and is usually bound to the surface of the microorganism or present in the culture solution. As the cellulosome, any one of cellulosomes produced by known cellulosome-producing microorganisms, cellulosomes to be clarified in the future, and modified forms thereof such as anaerobic microorganisms including microorganisms that form cellulosomes shown in Table 6 can be used. It can be used for the first scaffold protein of the invention and the second scaffold protein described below. In view of the high cellulose-degrading ability and the like, cellulosomes produced by thermophilic anaerobic microorganisms such as clostridium thermocellum, clostridium bacteria such as clostridium cellulolyticum, or modified versions thereof can be used in the present invention.

(First scaffold protein)
The first skeletal protein is basically a protein composed of a single polypeptide chain and can have the following domains. Hereinafter, these domains will be described.

(Foreign protein binding domain)
The foreign protein binding domain is a domain for binding and holding cellulase, which is a foreign protein for yeast. This domain is derived from the type I cohesin domain of the cellulosomal type I scaffoldin protein. The type I cohesin domain of the scaffolding protein is a noncovalent bond of the cellulase having catalytic activity in the type I scaffolding protein of the cellulosome (cellulase complex) formed by various cellulosome-producing microorganisms shown in Table 6. It is known as a binding domain (Nakaku et al., Protein Nucleic Acid Enzyme, Vol.44, No.10 (1999), p41-p50, Demain, AL, et al., Microbiol Mol. Biol Rev., 69 (1 ), 124-54 (2005), Doi, RH, et al., J. Bacterol., 185 (20), 5907-5914 (2003), etc.). Many of such type I cohesin domains have been sequenced in various cellulosome-producing microorganisms. The amino acid sequences and DNA sequences of these various type I cohesins are various protein databases and DNA sequence databases accessible via NCBI HP (http://www.ncbi.nlm.nih.gov/), etc. Can be obtained more easily.

  Examples of the foreign protein binding domain of the first skeletal protein include type I cohesins such as Clostridium thermocellum type I scaffolding protein, Clostridium josui type I scaffolding protein, etc. Domains can be used. In addition, C.cellulolyticum (NCBI website http://www.ncbi.nlm.nih.gov/), accession number: U40345), C.cellulovorans (same website, accession number: M73817), C.acetobutylicum ( The type I cohesin domain disclosed in the homepage, accession number: AE001437) is also included.

  In the present specification, the expression “derived from a predetermined domain of a protein” means comprising an amino acid sequence of the predetermined domain existing in nature or a modified sequence thereof. The modified sequence is a sequence in which the amino acid sequence is modified within a range that exhibits the same action as the predetermined domain. Such an amino acid sequence has, for example, preferably 90% or more, more preferably 95% or more, and still more preferably 99% or more identity with the amino acid sequence of the predetermined domain.

  The first scaffold protein preferably has a plurality of such foreign protein binding domains in tandem. The term “having tandem” is sufficient if a plurality of domains are aligned in the amino acid sequence. The presence or absence of a spacing sequence between the aligned protein binding domains, the spacing spacing being constant, Regardless of the existence of the domain. Preferably, there is a spaced arrangement between the domains. The plurality of foreign protein binding domains may be the same type of domain consisting of one type of amino acid sequence, or may be a domain consisting of two or more types of different amino acid sequences. By using different types of foreign protein binding domains, different foreign proteins can be arranged on the first scaffold protein.

  The first skeletal protein preferably has a foreign protein binding domain to such an extent that yeast acquires aggregability. When yeast is used while retaining proteins such as enzymes, if these yeasts can aggregate, enzymes can be aggregated on the agglomerated yeast, and yeast can be efficiently recovered and used repeatedly. be able to.

  In order to impart aggregability to yeast by the first skeletal protein, it is preferable to have three or more foreign protein binding domains. This is because if it is 3 or more, the yeast tends to be imparted with aggregability. Preferably it is 4 or more. It is because it becomes easy to give cohesiveness to yeast clearly that it is four or more. From the viewpoint of aggregation, the upper limit of the number of foreign protein binding domains is not particularly limited. In addition, from the viewpoint of easily ensuring the expression level, it may be preferable that the number is about 7 or less.

  The foreign protein binding domain can bind and retain the foreign protein with a binding property based on the type I cohesin domain. That is, the type I cohesin domain can bind to a foreign protein having a type I dockerin domain. The type I dockerin domain is a site that binds non-covalently to the type I cohesin of the type I scaffoldin protein, and is a domain that is included in the cellulase that constitutes the cellulosome. For example, when the foreign protein binding domain is derived from a type I cohesin derived from clostridium thermocellum, the type I dockerin domain includes the amino acid sequence represented by SEQ ID NO: 15. Clostridium cellulolyticum, Clostridium thermocello rovolans, Clostridium jossui, Clostridium acetobutylicum, Luminococcus flavefaciens (R. fravefaciens), Acid thermos cellulolyticus (A. cellulolyticus), Bacteroides cellosolvens ( When derived from a type I cohesin derived from B. cellulosolvens), the amino acid sequences represented by SEQ ID NO: 16 to SEQ ID NO: 22 are mentioned, respectively.

  The foreign protein binding domain only needs to be provided in the first skeletal protein where it does not interfere with the binding to the second skeletal protein. Typically, it is provided at a site other than the N-terminus.

(Second scaffold protein binding domain)
The second scaffold protein binding domain is a domain for binding the first scaffold protein to the second scaffold protein. This domain is known as a domain for retaining a type I skeletal protein on a type II skeletal protein non-covalently in cellulosomes (Koukan et al., Protein Nucleic Acid Enzyme, Vol. 44, No. 10 (1999). ), P41-p50 etc.). When only the first skeletal protein is displayed on the surface layer of yeast, the first skeletal protein may not have the second skeletal protein binding domain.

  A large number of type II dockerin domains have been determined in various cellulosome-producing microorganisms. The amino acid sequences and DNA sequences of these various type II dockerin domains are the various protein databases and DNA sequences accessible via NCBI's HP (http://www.ncbi.nlm.nih.gov/), etc. It can be easily obtained from the database. For example, the type II dockerin domain of clostridium thermocellum includes the amino acid sequence set forth in SEQ ID NO: 23, and the same domains of acid thermos cellulolyticus and luminococcus flavfaciens include SEQ ID NO: 24 and The amino acid sequence described in 25 is mentioned.

  Since the second skeletal protein binding domain is a domain that binds to the second skeletal protein binding domain, the second skeletal protein can be bound to the C terminus or N terminus of the first skeletal protein or in the vicinity thereof. Provided. Typically, it is provided at or near the C-terminus of the first scaffold protein.

(A domain consisting of the amino acid sequence represented by SEQ ID NO: 26 or an amino acid sequence having 90% or more identity with the amino acid sequence)
By providing this domain (hereinafter referred to as the third domain in the present specification), the activity of the foreign protein can be enhanced as a result, compared to yeast that does not have the third domain. The effect of the third domain on enhancing the activity of the foreign protein as a result is not always clear as described above. The amino acid sequence represented by SEQ ID NO: 26 is a sequence called an X module adjacent to a type II dockrin of a type I scaffoldin protein of clostridium thermocellum. The function of the X module is not always clear, and it is quite known how it works when used for the first skeletal protein that is not clostridium thermocellum or genus clostridium and is retained on the surface of fungal yeast cells. Not.

  As such a third domain, in addition to the amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence having 90% or more identity with the amino acid sequence can be used. More preferably, it is 95% or more. That is, the amino acid sequence represented by SEQ ID NO: 26 may comprise an amino acid sequence having one or more mutations selected from substitution, deletion, insertion and addition of one or several amino acids. . Preferably, it is 1 or more and 10 or less, more preferably 1 or more and 5 or less mutations, and further preferably 1 or more and 3 or less.

  According to the disclosure of the present specification, the third domain that can be used in the present invention is limited to an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 26 as described above. It is not necessary to be derived from the same strain or species of type I scaffolding protein X module from the cellulosome-producing microorganism from which the second scaffold protein binding domain is derived, but different strains and other species of the same species Although it may be derived from the cellulosome-producing microorganism, it is preferably derived from the X module of the same species or strain.

  As the amino acid sequence of the third domain derived from other than clostridium thermocellum which is the origin of the amino acid sequence represented by SEQ ID NO: 26, for example, the amino acid sequence represented by SEQ ID NO: 27, which is an X module derived from Acid thermos cellulolyticus, is used. Amino acid sequence (identity with SEQ ID NO: 26 is 62%), amino acid sequence represented by SEQ ID NO: 28, which is an X module derived from Luminococcus flavus faciens (identity with SEQ ID NO: 26) Is 21%).

  The first skeletal protein has a third domain in the vicinity of the second skeletal protein binding domain, preferably adjacent to the second skeletal protein binding domain and in the middle of the end where the second skeletal protein binding domain is present. Prepare for more parts. When the second scaffold protein binding domain is on the C-terminal side, it is preferably provided adjacent to the N-terminal side of the second scaffold protein binding domain.

(Cellulose binding domain)
When the foreign protein-retaining yeast is used for cellulose degradation, the first skeletal protein may have a cellulose-binding domain derived from a cellulose-binding domain (CBD) of a cellulosome type I scaffoldin protein. preferable. The cellulose-binding domain is known as a domain that binds to cellulose, which is a substrate in the type I scaffolding protein (supra Crown). One or two or more cellulose-binding domains may be provided. Note that the cellulose-binding domain is not required in all of the first skeletal proteins provided in yeast.

  Many of the amino acid sequences and DNA sequences of CBDs in the cellulosomes of various cellulosome-producing microorganisms have been determined, and the amino acid sequences and DNA sequences of these various CBDs can be obtained from NCBI HP (http: //www.ncbi.nlm. It can be easily obtained from various protein databases and DNA sequence databases accessible via nih.gov/). For example, the cellulose binding domain of clostridium thermocellum includes the amino acid sequence represented by SEQ ID NO: 29. In addition, as the cellulose binding domain of Clostridium cellulolyticum, Clostridium cellulovolans, Clostridium acetobutylicum, Clostridium jossii, Acid thermos cellulolyticus, Bacteroides cellulovorans, the amino acid sequences represented by SEQ ID NOs: 30 to 35, respectively, The domain which has is mentioned.

  In the first skeletal protein, the cellulose-binding domain is preferably provided at the N-terminal, in the vicinity thereof, or in the entire length thereof more than the N-terminal.

  The first skeletal protein described above may be an artificial protein obtained by artificially combining the elements, or may be derived from a type I scaffoldin protein of a cellulosome-producing microorganism. That is, it may be the whole or a part of the type I scaffoldin protein or a modified form thereof. The modified body refers to a substance in which the whole or a part of the base sequence and / or amino acid sequence of the type I scaffoldin protein obtained from the cellulosome of the cellulosome-producing microorganism is at least partially modified. The first skeletal protein is a Clostridium thermocellum Type I Scaffoldin protein, Clostridium josui Type I Scaffoldin protein, all or a part thereof, or a modification thereof. Can do.

  The first skeletal protein can be presented on the cell surface of the yeast using the various known yeast surface layer display systems already described.

(Second scaffold protein)
The first skeletal protein may be presented on the cell surface by non-covalent bonding with a second skeletal protein bound to the cell surface. The second scaffold protein is a protein that has a first scaffold protein binding domain and is basically composed of a single polypeptide chain. The first skeletal protein binding domain is a domain that can bind the first skeletal protein non-covalently based on its amino acid sequence. Since the first skeletal protein can be bound non-covalently to the second skeletal protein, the first skeletal protein can be easily bound to the second skeletal protein or from the second skeletal protein. It can be easily separated and recovered.

(First skeletal protein binding domain)
As the first skeletal protein binding domain, a domain derived from a type II cohesin domain of a cellulosome type II scaffoldin protein (also referred to as an anchor protein) can be used. The type II cohesin domain is known as a domain for retaining a type I scaffoldin protein noncovalently on a type II scaffoldin protein in the cellulosome (Koukan et al., Protein Nucleic Acid Enzyme, Vol. 44, No. 10 (1999), p41-p50, etc.). It is known that the type II dockins domain of a type I scaffoldin protein binds to the type II cohesin domain of a type II scaffoldin protein.

  A large number of type II dockrin domains have been determined in various cellulosome-producing microorganisms. The amino acid sequences and DNA sequences of these various type II cohesins are various protein databases and DNA sequence databases accessible via NCBI HP (http://www.ncbi.nlm.nih.gov/), etc. Can be obtained more easily.

  As the first skeletal protein binding domain, for example, a type II cohesin domain of SdbA, Orf2, OlpB, which is a type II cohesin of clostridium thermocellum, can be used. Further, Sca D and Sca B type II cohesin domains of Acid thermos cellulolyticus, Sca A type II cohesin domain of Bacteroides cellosolvens, and the like can be mentioned.

  The second scaffold protein may have only one first scaffold protein binding domain, but a plurality of second scaffold proteins may be provided in tandem. By providing a plurality, it becomes easier to hold the cellulase on the surface of the yeast at a higher density.

  The plurality of first skeletal protein binding domains may be the same type of domain consisting of one type of amino acid sequence, or may be a domain consisting of two or more types of different amino acid sequences. By using different types of scaffold protein binding domains, different first scaffold proteins can be arranged on the second scaffold protein.

  The second scaffold protein needs to be bound to the surface side of the cell. The second scaffold protein is bound to the surface layer of the cell by a covalent bond or a non-covalent bond. The second scaffold protein has a yeast surface-binding domain for binding to the yeast surface layer.

  As a system for binding the second skeletal protein to the yeast surface side, various known yeast surface layer display systems described above can be used.

  Such a second skeletal protein may be constructed as a fusion protein obtained by artificially combining the first skeletal protein binding domain and the yeast cell surface binding domain described above, or a type II skeleton of a cellulosome-producing microorganism. It may be derived from protein. That is, it may be a part or all of the backbone protein or a modified form thereof. As such a type II skeletal protein, a clostridium thermocellum type II skeletal protein, a clostridium jossi type II skeletal protein, or a variant thereof can be used.

  There are several techniques for presenting the first skeletal protein to the cell surface via the second skeletal protein. One is that the first skeletal protein and the second skeletal protein are expressed in yeast, the second skeletal protein is presented on the cell surface layer, and the secreted first skeletal protein may be bound thereto. . Alternatively, only the second skeletal protein may be expressed and presented on the cell surface layer, and the first skeletal protein may be supplied from outside the cell and bound.

In addition, the first skeletal protein may be secreted and expressed in yeast, and the second skeletal protein may be prepared outside the yeast and supplied from the outside. In this case, it is preferable that the second scaffold protein has a cell surface binding domain based on the cell surface display system such as agglutinin described above. Furthermore, as another form, both the first skeletal protein and the second skeletal protein may be prepared outside the yeast and supplied to the yeast.
In this case, the second scaffold protein preferably has a cell surface binding domain based on a cell surface display system such as agglutinin.

  In order to retain cellulase with respect to such a first skeletal protein, cellulase can be secreted and produced extracellularly in yeast, and the cellulase can be retained by supplying it to the first skeletal protein. Alternatively, the cellulase produced outside the yeast may be brought into contact with the yeast that displays the first skeletal protein on the surface, so that the extracellularly produced cellulase is retained in the first skeletal protein. In order to retain the extracellularly produced cellulase on the first skeletal protein displayed on the surface, both may be brought into contact with each other in an appropriate aqueous medium. The aqueous medium is not particularly limited as long as the physiological activity of yeast and cellulase is maintained. For example, appropriate pH, osmotic pressure and temperature may be ensured for these. The pH varies depending on proteins and cells, but is usually in the range of pH 6 to 9. The osmotic pressure is preferably approximately isotonic with respect to cells such as microorganisms. The osmotic pressure can be adjusted by using an appropriate salt in addition to a buffer solution and an isotonizing agent. Further, the temperature is preferably 1 ° C. or higher and 10 ° C. or lower, more preferably 2 ° C. or higher and 5 ° C. or lower in consideration of protein denaturation and the like. Such an aqueous medium may be a salt solution or the like. For example, a Tris-HCl buffer of about 20 mM to 50 mM may be used. Moreover, it is good also as an aqueous medium in which calcium ion exists, such as CaCl2 solution more than 0 mM and 20 mM or less, More preferably, 5 mM or more and 15 mM or less. More preferably, it is an aqueous medium in which about 10 mM calcium ions are present. If necessary, the aqueous medium may be agitated to promote contact between the two.

  In addition, those skilled in the art appropriately determine the amino acid sequence and base sequence of the cellulase gene, and the amino acid sequence and base sequence of the first skeletal protein and the second skeletal protein, and hold each of these genes so that they can be expressed. These proteins can be obtained by using a recombinant vector. In addition, these various proteins may be synthesized outside the yeast of the present invention using a protein expression system utilizing Escherichia coli, yeast or the like, or secreted and expressed in the yeast of the present invention, or the yeast of the present invention. The yeast of the present invention can be obtained by presenting the cell surface. General operations necessary for the production of such a recombinant vector and the handling of yeast or the like as a recombinant host are those commonly performed by those skilled in the art. A person skilled in the art can carry out this method by referring appropriately to an experimental book by Maniatis, J. Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, 1989, 2001). Various operations for expressing foreign proteins by introducing such genes into various cells including yeast should follow protocols such as Molecular Cloning A Laboratory Manual second edition (Maniatis et al., Cold Spring Harbor Laboratory press. 1989). it can. In addition, as a method for introducing a vector, various conventionally known methods such as calcium phosphate method, transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method or other methods Is mentioned. Such a method is described in the above-mentioned experiment document. Yeast that expresses a necessary protein can be obtained by selection using a marker gene and selection based on expression of the yeast into which the vector has been introduced.

(yeast)
The yeast that holds the cellulase selected from the cellulase group on the cell surface layer is not particularly limited, and various known yeasts can be used. Saccharomyces cerevisiae and other Saccharomyces yeasts, Schizosaccharomyces pombe Schizosaccharomyces pombe yeasts, Candida krusei, Candida shehatae, etc. Candida yeast, Pichia pastoris, Pichia stipitis, Pichia yeast, Hansenula yeast, Trichosporon yeast, Brettanomyces genus Examples include yeasts, yeasts of the genus Pachysolen, yeasts of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis. It is done. Of these, Saccharomyces yeasts are preferable from the viewpoint of industrial availability and the like, and Saccharomyces cerevisiae is more preferable.

  In addition, the yeast of this invention or its combination may express (beta) -glucosidase extracellularly with the cellulase selected from the cellulase group of this invention. In addition, the yeast combination of the present invention may include a yeast that produces β-glucosidase extracellularly. By expressing β-glucosidase outside the cell, the cellulose degradation product obtained by the cellulase selected from the cellulase group of the present invention can be effectively degraded to glucose. In addition, useful substances can be produced directly by assimilating glucose derived from the cellulase. As a form which produces (beta) -glucosidase outside a cell, the form secreted and produced outside a cell and the form hold | maintained on a cell surface layer similarly to the cellulase group of this invention are mentioned. For the secretory production of proteins in yeast, a signal peptide that functions in yeast may be added. For example, signal peptide sequences derived from yeast include, for example, yeast invertase leader, alpha factor leader, Rhizopus oryzae and C. albicans glucoamylase leader. In addition, in order to make yeast produce β-glucosidase, the transformation method by gene introduction already described can be used.

  As the yeast, yeast that produces an organic acid such as lactic acid or a compound that also serves as an industrial raw material such as C3 to C5 alcohol by genetic engineering modification can be used. By making β-glucosidase coexist in the degradation system and assimilating glucose derived from cellulose in such yeast, useful substances can be directly produced using cellulose as a raw material. For example, transformed yeasts such as lactic acid-producing yeast are disclosed in, for example, JP-A-2003-334092, JP-A-2004-187743, JP-A-2005-137306, JP-A-2006-6271, JP-A-2006-20602, JP-A-2006-42719. JP-A-2006-75133, JP-A-2006-296377, etc., and these transformed yeasts can be used in the present invention. All of the contents described in these publications are incorporated herein by reference.

  As described above, the yeast of the present invention or a combination thereof retains the cellulase capable of exhibiting a good cellulase activity on the cell surface of the yeast and the combination thereof on the cell surface. For this reason, the cellulose in a cellulose containing material can be decomposed | disassembled efficiently by making the yeast of this invention and a cellulose containing material contact. In particular, by introducing such a cellulase gene into yeast and expressing it, a preferred cellulase can be obtained immediately by culturing the yeast. Moreover, when yeast decomposes | disassembles cellulose and can utilize and proliferate cellulose, the useful substance production by the enhancement of a further cellulose decomposition activity and fermentation will be attained.

(Method for Producing Cellulose Degradation Product) The method for producing a cellulose degradation product of the present invention is a method for producing a cellulose degradation product from cellulose using cellulase. The production method of the present invention can comprise a step of degrading cellulose with two or more cellulases selected from the cellulase group using the yeast of the present invention or a combination thereof. According to the present invention, cellulose can be efficiently decomposed and reduced in molecular weight to produce cellulose oligomers, cellobiose or glucose.

  In order to decompose cellulose with two or more types of cellulases selected from the group of cellulases of the present invention, a yeast that simultaneously holds two or more types of cellulases may be used, or a yeast that holds one type of cellulase on the cell surface layer. May be used in combination of two or more, or such yeasts may be used in combination. When cellulase is retained on the surface of the yeast cell via the first skeletal protein derived from cellulosome, the yeast tends to aggregate with each other. For this reason, even if two or more types of cellulases are held in the cell surface layer of different yeasts, cellulose can be efficiently decomposed. In addition, when the first skeletal protein has a cellulose-binding domain, yeast is easily adsorbed to cellulose. Therefore, even if two or more cellulases are held in different yeasts, the efficiency is high. Cellulose can be decomposed. In addition, as already explained, the combination of cellulases depends on the difference in crystallinity (crystalline or amorphous) of the cellulose to be decomposed, the degree of crystallinity, and the blending ratio of different crystalline celluloses. It can also be selected as appropriate.

  From the viewpoint of obtaining glucose as a degradation product, β-glucosidase is preferably present in the degradation step. β-glucosidase may be added to the decomposition step as an enzyme agent. In addition, β-glucosidase may be provided by a yeast that produces β-glucosidase extracellularly together with a cellulase selected from the cellulase group of the present invention, or may be provided by a yeast that expresses β-glucosidase.

  The decomposition step is performed by bringing the yeast of the present invention into contact with the cellulose-containing material. Although the aspect of decomposition | disassembly is not specifically limited, It is preferable to make the yeast containing sufficient amount prepared beforehand contact a cellulose containing material, or to contact a cellulose containing material on the conditions which a yeast can proliferate. The conditions under which yeast can grow typically include conditions in which a medium containing a nutrient source for yeast growth is present. Among the nutrient sources, examples of the carbon source include glucose and sucrose which are easily used by yeast. In addition, when β-glucosidase is supplied to the decomposition step, yeast can be used using cellulose-derived glucose as a carbon source, and therefore it is not always necessary to add glucose or sucrose.

  In addition, the conditions in the decomposition step are determined in consideration of the survival, growth or fermentation conditions of the yeast of the present invention, the decomposition activity of cellulase retained on the surface layer, and the like.

  In the present specification, cellulose refers to a polymer in which glucose is polymerized by β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose in cellulose is not particularly limited, but is preferably 200 or more. In addition, examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. Cellulose may contain cellooligosaccharide and cellobiose, which are partially decomposed products thereof. Further, the cellulose may be crystalline cellulose or non-crystalline cellulose, but preferably contains crystalline cellulose. Furthermore, the cellulose may be naturally derived or artificially synthesized. The origin of cellulose is not particularly limited. It may be derived from plants, fungi, or bacteria.

  Cellulose may be subjected to a decomposition step in the form of a cellulose-containing material. In the present specification, the cellulose-containing material may be any material that contains cellulose as described above. Therefore, it may consist essentially of cellulose, and the cellulose-containing material may be composed of glycoside β-glucoside, lignin and / or lignocellulose which is a complex with hemicellulose, and pectin. It may be a complex. Cellulose-containing materials include natural fiber products such as cotton and hemp, recycled fiber products such as rayon, cupra, acetate, and lyocell, various straws such as rice straw, agricultural waste such as rice husks, bagasse and wood chips, and waste paper And biomass (woody and herbaceous) containing various wastes such as building waste.

  Depending on the type of cellulose-containing material to be degraded, pretreatment or additional enzymes may be required. For example, when using lignocellulosic materials, physical, chemical or enzymatic pretreatment may be required for efficient degradation.

  Various cellulose degradation products are obtained by carrying out the degradation step. In order to recover the degradation product, the culture supernatant may be recovered. The culture supernatant can be used as various carbon sources as it is or after being appropriately purified.

(Useful substance production method)
The method for producing a useful substance of the present invention is a method for producing a useful substance from cellulose, wherein two or more cellulases selected from the cellulase group of the present invention using one or more of the yeasts of the present invention. And a step of producing a useful substance by the yeast or other microorganisms in the presence of a carbon source containing a cellulose degradation product obtained by degrading cellulose. According to the method for producing a useful substance of the present invention, cellulose, which is a typical material of plant biomass, can be produced in a form that can be used by microorganisms, and the use of petroleum resources can be suppressed or avoided to produce a useful substance.

  In the production process of useful substances, any microorganism that can use glucose, such as Escherichia coli, Bacillus subtilis, Neisseria gonorrhoeae, etc., can be used in addition to yeast. Examples of microorganisms that can use glucose include those that have been modified to produce compounds that are not the original metabolites by replacing or adding one or more enzymes in the metabolic system from glucose by genetic recombination. There may be.

  The production process of the useful substance is not particularly limited as long as the cellulose degradation product by the yeast of the present invention is used, and various forms can be adopted. For example, the useful substance production process can take various forms in relation to the cellulose decomposition process by the yeast of the present invention. For example, in one form, a cellulose decomposition process and a useful substance production process are implemented substantially as one process. That is, in the cellulose degradation step, when β-glucosidase is present in addition to the yeast of the present invention, the degradation product is glucose, so that the yeast of the present invention assimilate the glucose derived from cellulose to produce a useful substance. can do.

  Moreover, in another one form, a useful substance production process is implemented after the cellulose decomposition process by the yeast of this invention. That is, when β-glucosidase is not present in the cellulose decomposition step, β-glucosidase is supplied to the culture supernatant containing the cellulose degradation product obtained in the decomposition step and decomposed to glucose, or decomposed. In addition, there may be mentioned a form in which the step of producing useful substances by assimilating glucose with the yeast of the present invention or other yeasts or other microorganisms is mentioned. Note that β-glucosidase in this form may be supplied in any form such as an enzyme preparation, extracellular production of yeast or microorganisms, and the like.

  Even when β-glucosidase is contained in the cellulose decomposition step, a useful substance production step is performed in which the decomposition product containing glucose obtained in the decomposition step is used as a carbon source for other yeasts or microorganisms. It is also possible.

  In the production process of useful substances, culture conditions generally applied to yeast or the microorganism to be used can be appropriately selected and used. Typically, stationary culture, shaking culture, aeration-agitation culture, or the like can be used as the culture method. The aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. The culture temperature is also appropriately determined according to the type of microorganism used, and is not particularly limited, but can be in the range of 25 ° C to 55 ° C. Moreover, although culture | cultivation time is also set as needed, it can be set as several hours-about 150 hours. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary.

  Although it does not specifically limit as a useful substance, What is necessary is just what yeast and other microorganisms can produce using glucose. The useful substance may be a compound that is not an original metabolite by replacing or adding one or more enzymes in the metabolic system from glucose in yeast by genetic recombination. Specifically, in addition to lower alcohols such as ethanol, organic acids such as lactic acid, fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.) by adding an isoprenod synthesis route, glycerin by modifying glycolysis, plastics and chemical raw materials For example, materials targeted by biorefinery technology.

  By performing the production process of the useful substance, the useful substance is produced according to the useful substance production capacity of the used microorganism. For example, since general yeast ferments ethanol, ethanol can be obtained. Yeast having the ability to produce an organic acid such as lactic acid by genetic engineering modification or the like produces lactic acid or the like. After completion of the useful substance production step, a step of recovering a useful substance-containing fraction from the culture solution, and a step of purifying or concentrating the fraction can also be performed. The recovery process and the purification process are appropriately selected according to the type of useful substance.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples and can be implemented in various forms without departing from the gist of the present invention. can do. In addition, the gene recombination operation described below was basically performed according to Molecular Cloning: A Laboratory Manual (T. Maniatis, et al., Cold Spring Harbor Laboratory).

  C. thermocellum (ATCC27405) was obtained from ATCC, sterilized water was directly added to the dried cells, suspended, and then subjected to genome extraction with DNeasy Kit (manufactured by QUIAGEN). The cellulase gene excluding the signal peptide from the C. thermocellum genome was cloned, inserted into the 2 μm vector shown in FIG. 1, and then introduced into S. cerevisiae MT8-2. The cloned enzyme is shown in Table 7. Each enzyme was cultured overnight in YPD, and the supernatant was subjected to a halo assay using carboxymethylcellulose (CMC). CelA, CelD, CelF, CelR, CelK, and CbhA showed activity. For Family 48 cellulase CelS, which has only processive or exoglucanase activity, the yeast supernatant was mixed with 50 mM acetate buffer pH 6.O 0.5% phosphate-swelled cellulose (PSC), and the amount of free reducing sugar was measured by TZ assay. However, no activity was detected.

  AGA1 gene is introduced into the genome of S. cerevisiae BJ5465, and DNA corresponding to an amino acid sequence containing a cellulose binding domain (CBD) +4 cohesin domain, which is a part of the C. thermocellum-derived scaffoldin protein (SEQ ID NO: 36 The DNA was fused with the Aga2 protein and introduced into the genome of the same strain to obtain BJ004 strain. Furthermore, A. aquleatus-derived β-glucosidase (BGL) gene was introduced into the genome to obtain BJ104 strain.

From the results of Example 1, it is known that the cloned cellulase derived from C. thermocellum is expressed in S. cerevisiae except for CelS. Therefore, 2 μm vector C. thermocellum-derived CelA, CelD, CelR, CelF, CelK, and CbhA used in Example 1 were introduced into the BJ104 strain to prepare BJ104pA, BJ104pD, BJ104pO, BJ104pR, and BJ104pCb yeasts. Prepare each yeast to OD600 = 5.1 ml, wash twice with 1 ml of sterilized water + 10 mM CaCl ₂ solution, suspend in 1 ml of 1% CMC 50 mM acetate buffer pH 6.0, and perform CMC degradation test at 60 ° C for 2 hours. And the reducing sugar activity was measured by TZ assay. The results are shown in FIG. As shown in FIG. 2, each yeast exhibited activity.

  The yeast prepared in Example 2 was prepared to D600 = 10 alone and in combination of two kinds, and a 0.5% PSC degradation test was conducted at 50 ° C. for 45 hours. The results are shown in FIG. As shown in FIG. 3, a synergistic effect was observed when EG (CelA, CelD) was combined with a processing enzyme (CelF, CelR), and in particular, a processing endoglucanase belonging to GH family 9, CelR and other enzymes were combined. A high synergistic effect was observed when combined.

  FIG. 4 shows the results of preparing the yeast prepared in Example 2 alone and in combination of two kinds to D600 = 10 and conducting a 1% Avicel degradation test at 50 ° C. for 72 hours. As shown in FIG. 4, a high synergistic effect was observed when the combination containing a processing enzyme (CelR, CelK, CbhA) belonging to GHF9 was used. In addition to CelA and CelF, CelD also showed a good synergistic effect as the enzyme to be combined.

  In this example, two types of enzymes were co-expressed in one yeast to evaluate suitable enzyme combinations for the degradation of crystalline cellulose.

The Trp1 gene of yeast BJ104 was disrupted to produce a Trp-requiring strain. G418 was used as a marker, and a Trp homologous recombination region was added to both ends thereof for PCR. We won ^- this PCR fragment introduced Trp auxotrophs BJ104T yeast BJ104. Cellulase was introduced into pRS436SSRG (Ura3 marker) and pRS434SSRG (Trp1 marker) with a 2μm plasmid in this strain. . After collection, OD600 was set to 10 and washed twice with DW + 10 mM CaCl2, and then reacted with 50 mM acetate buffer pH 6.0, 1% Avicel at 50 ° C. for 80 hours. The results are shown in FIG.

  As shown in FIG. 5, high degradation activity was observed when Family 9 processing enzymes were combined (R-K, R-CbhA, K-CbhA). In particular, the combination of K-CbhA showed the highest activity. When A-R, A-K, and A-Cb were expressed in the same yeast, the activity was lower than the combination of yeasts expressing a single enzyme. In yeast, the expression of CelA was remarkably higher than that of other enzymes, and it was thought that most of them would have CelA activity when produced in the same yeast.

In this embodiment, K-CBHA-expressing yeast BJ104T with the highest activity produced in Example 5 ^- PKCb similarly cultured as in Example 5 (SD-Ura, after overnight incubation at Trp, 72 hours SD-Ura, After culturing with Trp + 2% CAA), OD600 = 40 and YP + 10% Avicel was cultured at 30 ° C. for 65 hours. The results of measuring the ethanol concentration in the medium are shown in FIG. As shown in FIG. 6, K-CBHA expression yeast BJ104T ^- pKCb was detected 0.24% ethanol in medium YP + 10% Avicel. In the case of YP alone, 0.09% ethanol was detected. From the above, it was found that K-CbhA-expressing yeast BJ104T ^- pKCb was saccharified and ethanol-fermented with about 3% of Avicel.

Claims (14)

  Cellobiohydrolase Cel 5F, Endoglucanase Cel 5C belonging to Glycolytic Enzyme Family 5, Endoglucanase Cel 8A belonging to Glycolytic Enzyme Family 8, Endoglucanase Cel 9D belonging to Glycolytic Enzyme Family 9, Endoglucanase Cel A yeast or a combination thereof that retains one or more cellulases selected from the group consisting of 9R, cellobiohydrolase Cel 9K, and cellobiohydrolase Cbh A on the cell surface.   The yeast or a combination thereof according to claim 1, wherein the cellulase retained in the cell surface of the yeast is at least two kinds including at least a processive cellulase.   The yeast or combination thereof according to claim 1 or 2, wherein the cellulase retained in the cell surface layer of the yeast is at least two kinds including cellulases belonging to the saccharide-degrading enzyme family 9. The cellulase retained in the cell surface layer of the yeast comprises cellulase shown in the first column of the following table and one or more selected from the cellulase described in the second column corresponding to the first column. The yeast according to any one of claims 1 to 3, or a combination thereof.
The cellulase retained in the cell surface layer of the yeast comprises cellulase shown in the first column of the following table and one or more selected from the cellulase described in the second column corresponding to the first column. The yeast according to any one of claims 1 to 4, or a combination thereof.
The cellulase retained in the cell surface layer of the yeast has the following relationship with the base sequence or amino acid sequence represented by the sequence numbers shown in the following table, respectively. Yeast or a combination thereof.
(A) Cellulase encoded by a gene comprising a base sequence represented by each sequence number described in Table 10 (b) DNA complementary to DNA comprising a base sequence represented by each sequence number described in Table 10 A cellulase encoded by a gene that hybridizes under stringent conditions with cellulase activity (c) a base sequence having a homology of 80% or more to the base sequence represented by each SEQ ID No. in Table 10 A cellulase having a corresponding cellulase activity encoded by a gene consisting of: (d) a cellulase having an amino acid sequence represented by each SEQ ID NO: listed in Table 10 (e) an amino acid sequence represented by each SEQ ID NO: listed in Table 10 In which one or more amino acids are deleted, substituted or added and have a corresponding cellulase activity Cellulase with corresponding cellulase activity has an amino acid sequence having 80% or more identity to the amino acid sequence represented by the SEQ ID NO: according to cellulase (f) Table 10
  The yeast or a combination thereof according to any one of claims 1 to 6, wherein the cellulase retained in the cell surface layer of the yeast is derived from Clostridium.   The yeast or a combination thereof according to claim 7, wherein the cellulase is derived from Clostridium thermocellum.   The yeast according to any one of claims 1 to 8, wherein the cellulase retained in the cell surface layer of the yeast is retained in the cell surface layer via a protein derived from a scaholdin protein of a cellulosome-producing microorganism. combination.   The yeast according to claim 9 or a combination thereof, wherein the protein derived from the scaffolding protein is derived from a scaffolding protein of Clostridium thermocellum.   The yeast or a combination thereof according to claim 9 or 10, wherein the protein derived from the scaffolding protein is derived from a type I scaffolding protein.   The yeast or a combination thereof according to any one of claims 1 to 11, wherein the yeast or a combination thereof includes a yeast secreting and producing β-glucosidase. A method for producing a cellulose degradation product from cellulose using cellulase,
A step of decomposing cellulose using two or more cellulases held by the yeast or a combination thereof using the yeast according to any one of claims 1 to 12 or a combination thereof.
A production method comprising: A method for producing useful substances from cellulose,
Carbon containing a cellulose degradation product obtained by degrading cellulose using two or more kinds of cellulases held by the yeast or a combination thereof using the yeast according to any one of claims 1 to 12 or a combination thereof. Producing a useful substance by the yeast or other microorganisms using a source;
A production method comprising: