JP6131764B2 - Artificial scaffold material and system for retaining proteins and use thereof - Google Patents

Description

  The present specification relates to artificial skeletal materials for retaining proteins and uses thereof.

  In order to use cellulose and hemicellulose, which are typical biomasses, an excellent cellulase that saccharifies (decomposes) these is required. There are many cellulases, and they are said to act in concert to improve their effects. Cellulase is a comprehensive concept of multiple types of enzymes that act on cellulose, such as endoglucanase, cellobiohydrolase, and β-glucosidase, and these cooperate to break down cellulose. These cooperative effects are events that are commonly observed in other enzymes.

  In order to perform high-efficiency cellulose saccharification with a plurality of cellulases, it is considered preferable to integrate and adjoin cellulases in order to impart saccharification ability. With regard to the accumulation of cellulose, attempts have been made to use cellulosomes formed by a complex of cellulase formed on the surface of bacterial cells and a backbone protein to which the cellulase binds. The backbone protein is a protein having a domain called cohesin, and is also referred to as a scaffolding protein. Cellulosomal cellulases have a domain called dockerin that binds cohesin. The backbone protein and cellulase are bound and complexed by non-covalent bond with cohesin-docklin.

  Attempts have also been made to artificially construct such natural cellulosomes (Patent Documents 1 and 2). In addition, the construction of cellulosome is promoted by modifying dockerin derived from clostridium thermocellum (Patent Document 3). Furthermore, attempts have been made to form cellulosomes derived from Clostridium cellulolyticum on the surface of yeast cells (Patent Document 4). Furthermore, a skeletal protein CipA derived from clostridium thermocellum, a skeletal protein CipC derived from clostridium cellulolyticum, and a skeletal protein derived from ScaB derived from luminococcus flavefaciens on the yeast surface are also produced. (Patent Document 5).

JP 2009-142260 A JP 2012-246922 A JP 2011-219399 A International Publication No. 2010/0956562 International Publication No. 2010/057064

  As described above, although various attempts have been made to construct artificial cellulosomes, expression of bacterial cellulosomes in eukaryotic microorganisms such as yeast is still insufficient to obtain the intended saccharification ability. there were. That is, although the construction of artificial cellulosomes in the cell surface of eukaryotic microorganisms has been developed, it did not reach the expected cellulolytic activity. That is, in order to artificially reconstruct a structure in a heterogeneous organism on a eukaryotic microorganism, further optimization of the cellulosome to the eukaryotic microorganism was necessary.

  The present specification provides a more suitable artificial scaffold protein system and its use for constructing artificial cellulosomes on eukaryotic microorganisms.

  The present inventors pay attention to the cellulosome of luminococcus flavefaciens (hereinafter referred to as R. flavefaciens) among the cellulosomes of various bacteria, and the type III skeletal protein possessed by R. flavefaciens is a eukaryotic microorganism. It was found to be suitable for production and surface display. In addition to examining the number of cohesins in type III skeletal proteins, we improved dockerin binding strength for binding cellulase to the skeletal proteins, and examined dockerin binding sites in cellulases, each of which is an artificial cellulosome via a eukaryotic microorganism. We obtained useful knowledge about the construction of The present specification provides the following means based on these findings.

(1) An artificial skeletal material for holding one or more target proteins,
An artificial skeletal material comprising a protein having 1 or more and 20 or less cohesins derived from a type III cohesin and having a binding activity with a type III dockin as a holding site for the one or more target proteins.
(2) The artificial skeletal material according to (1), comprising three or more cohesins.
(3) The artificial skeletal material according to (1) or (2), comprising six or more cohesins.
(4) The artificial skeleton material according to any one of (1) to (3), comprising eight or more cohesins.
(5) The artificial skeletal material according to any one of (1) to (4), wherein the cohesin has substitution in any one of the following modes at one or more N-type sugar chain modification sites.
(A) Substitution of asparagine to amino acid residues other than asparagine (b) Substitution of asparagine two residues downstream of serine or threonine to amino acid residues other than serine and threonine (6) The cohesin is derived from R. flavefaciens The artificial skeletal material according to any one of (1) to (5), which is a ScaB cohesin.
(7) The artificial skeletal material according to any one of (1) to (6), which is an artificial skeletal material that is expressed in a eukaryotic microorganism and presented on the cell surface.
(8) The artificial skeletal material according to (6), wherein the eukaryotic microorganism is yeast.
(9) the artificial skeletal material according to any one of (1) to (8);
One or more target proteins each derived from a type III dockerin and having a dockin binding activity to the cohesin;
A composite material comprising:
(10) The composite material according to (9), wherein the dockrin each of the one or more target proteins has substitution of any one of the following modes at one or more N-type sugar chain modification sites .
(A) Substitution of asparagine to an amino acid residue other than asparagine (b) Substitution of serine or threonine at two residues downstream of asparagine to amino acid residues other than serine and threonine (11) One or more target proteins Is a composite material according to (9) or (10), which is one or more enzymes.
(12) The composite material according to any one of (9) to (11), wherein the one or more target proteins are one or more cellulases.
(13) The composite according to any one of (9) to (12), wherein the one or more cellulases include a cellulase derived from Phanerochaete chrysosporium and / or a cellulase derived from Trichoderma reesei comprising the dockrin on the N-terminal side. material.
(14) The composite material according to any one of (9) to (13), wherein the one or more cellulases include a cellulase derived from Clostridium thermocellum having the dockrin on the C-terminal side.
(15) A eukaryotic microorganism having the artificial skeletal material according to any one of (1) to (8) on a cell surface layer.
(16) A eukaryotic microorganism having the composite material according to any one of (9) to (14) on a cell surface layer.
(17) A recombinant vector retaining DNA encoding the protein of the artificial skeletal material according to any one of (1) to (8).
(18) The composite material according to any one of (9) to (14), wherein the one or more target proteins contact the composite material containing one or two or more cellulases and the cellulose-containing material. A method for producing a saccharified product from a cellulose-containing material, comprising a step.
(19) The composite material according to any one of (9) to (14), wherein the one or more target proteins are cellulose in the presence of a composite material containing one or more cellulases and a cellulose-containing material. A method for producing a useful substance using a cellulose-containing material, comprising a step of culturing a eukaryotic microorganism that assimilate a saccharified product.
(20) A method for producing a useful substance using a cellulose-containing material, comprising a step of culturing a eukaryotic microorganism assimilating a cellulose saccharified product in the presence of the eukaryotic microorganism and the cellulose-containing material according to (16).

It is a figure which shows a cohesin genome introduction | transduction vector. It is a figure which shows a dockrin genome introduction | transduction vector. It is a figure which shows an AGA1 genome introduction | transduction vector. It is a figure which shows the yeast surface layer presentation amount of cohesin. It is a figure which shows the yeast surface layer display amount of a dockrin. It is a figure which shows evaluation of cohesin-docklin binding specificity. It is a figure which shows the surface layer display amount of yeast of RfCoh. It is a figure which shows a high copy number yeast secretion expression vector. It is a figure which shows a RfDoc fusion expression vector. It is a figure which shows the PSC decomposition | disassembly activity of RfDoc fused PcCBH2, TrEG2. It is a figure which shows the PSC decomposition | disassembly activity of various RfDoc fusion cellulases. It is a figure which shows the comparison result of PSC decomposition | disassembly activity of various RfDoc fusion cellulases in culture | cultivation temperature (20 degreeC, 30 degreeC). It is a figure which shows the amino acid sequence of RfDoc and the N-type sugar chain modification plan site. It is a figure which shows the modification scheme of RfDoc. It is a figure which shows a low copy number yeast secretory expression vector. It is a figure which shows the improvement of PSC decomposition | disassembly activity by modification | change of RfDoc. It is a figure which shows the sequence-analysis result after RfDoc modification. It is a figure which shows the amino acid sequence of Scab-4 cohesin and the N-type sugar chain modification plan site. It is a figure which shows the sugar_chain | carbohydrate removal scheme from ScaB-4 cohesin. It is a figure which shows a high copy number yeast secretion expression vector. It is a figure which shows the screening result of ScaB-4 cohesin after sugar chain removal. It is the figure 1 which shows the sequence-analysis result of sugar chain removal ScaB-4 cohesin. It is the figure 2 which shows the sequence-analysis result of sugar chain removal ScaB-4 cohesin. It is the figure 3 which shows the sequence-analysis result of sugar chain removal ScaB-4 cohesin. It is a figure which shows the cohesin surface layer presentation amount of a multiplex cohesin yeast. It is a figure which shows PSC degradation activity evaluation (cellulase simultaneous expression) using multi-cohesin yeast. It is a figure which shows Avicel degradation activity evaluation (cellulase simultaneous expression) using multi-cohesin yeast. It is a figure which shows PSC decomposition | disassembly activity evaluation (cellulase external addition) using multi-cohesin yeast. It is a figure which shows Avicel decomposition | disassembly activity evaluation (cellulase external addition) using multi-cohesin yeast. It is a figure which shows a cellulase genome introduction | transduction vector. It is a figure which shows a cellulase genome introduction | transduction vector. It is a figure which shows the comparison result of the Avicel decomposition | disassembly activity by the cohesin length of cellulosome display yeast. It is a figure which shows the N type sugar_chain | carbohydrate modification plan site in the X domain of ScaB. It is a figure which shows the binding evaluation result with ScaE cohesin with respect to X domain of sugar chain removal ScaB. It is a figure which shows the amino acid analysis result of sugar chain removal X domain. It is a figure which shows the relative activity when the PSC decomposition | disassembly activity of the cellulosome yeast which has natural type cohesin and dockrin is set to 1.

  The disclosure of the present specification relates to an artificial skeletal material that uses a cohesin-docklin bond to retain a target protein such as an enzyme and use thereof. According to the disclosure of the present specification, the functional expression of the target protein bound through the cohesin-docklin bond can be further improved by utilizing the type III cohesin-docklin bond and further controlling the number of the cohesins arranged. It turns out that it can be strengthened. Although not necessarily theoretically clear, the accumulation of the target protein is controlled by causing a eukaryotic microorganism to produce an artificial skeletal protein using a type III cohesin-docklin bond disclosed in the present specification. As a result, it is considered that the functionality of the target protein can be enhanced and the cooperative action of two or more target proteins can be enhanced. Cohesin (domain) is known as a domain that binds non-covalently to cellulase or other skeletal proteins with catalytic activity, which are provided in type I to III skeletal proteins in cellulosomes formed by cellulosome-producing microorganisms. (Kousaku 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.).

  For example, the present inventors can ensure the maximum amount of presentation by presenting a skeletal protein having about 4 type I cohesins on the surface of yeast cells, thereby agglutinating yeasts. An attempt was made to integrate the target protein (Japanese Patent Application No. 2007-199513). However, according to the disclosure of the present specification, although the amount of scaffold protein displayed decreases as the number of type III cohesins increases, contrary to expectation, it remains within a certain range as the number of cohesins increases. It was found that the functionality and cooperativity of the target protein improved.

  Hereinafter, the disclosure of the present specification will be described in detail with reference to various embodiments.

(Artificial skeleton material)
The artificial skeletal material disclosed in the present specification is an artificial skeletal material for holding one or more target proteins. The artificial skeletal material includes a protein having a cohesin derived from a type III cohesin and having a binding activity with a type III dockin (hereinafter also simply referred to as a type III cohesin) as a holding site for one or more target proteins. It is out. This artificial skeletal material retains the target protein based on the type III cohesin-docklin interaction. The cohesin-docklin interaction is non-covalent and is considered to be established by protein-protein interactions other than covalent bonds, such as hydrogen bonds, hydrophobic bonds, ionic bonds, and dipole interactions.

  This artificial skeletal material has a type III cohesin derived from a natural type III cohesin and having binding activity with a type III dockerin. Natural type III cohesins are phylogenetically classified based on the homology of the amino acid sequences of cellulosomal cohesins (domains) found in R. flavefaciens. Even with the highest similarity between R. flavefaciens ScaA and ScaB cohesins and those of the genus Clostridium, the similarity was less than 27% for certain ScaB cohesins. Moreover, although ScaA cohesin has a similarity of 85% or more to each other, it showed only a similarity of 25% or less with the ScaA cohesin of the genus Clostridium (Journal of Bacteriology, Vol.183, No.6, Mar. , 2001, p.1945-1953).

  Examples of type I cohesins include CipA derived from Clostridium thermocellum, CipC from Clostridium cellulolyticum, CipJ from Clostridium josui, CpbA from Clostridium cellulovorancs, and CipV from Acetivibrio cellulolyticus. Examples of type II cohesins include CipBC of Bacterioides cellulosolvens and Slp's which is an anchor protein of Clostridium thermocellum.

  Type III cohesin is originally retained in skeletal proteins such as ScaA, ScaB, ScaC, and ScaE of R. flavefaciens, which is a bacterium. When it is intended to be secreted, it is preferable from the viewpoint that the expression level is larger than that of cohesin, a skeletal protein derived from other bacteria. That is, it is more suitable for production by eukaryotic microorganisms than type I and II cohesins. In particular, ScaB type III cohesin is preferred. ScaB type III cohesin is excellent in extracellular secretion and cell surface presentation.

  Examples of such natural type III cohesins include cohesins in the ScaA, ScaB, ScaC, and ScaE backbone proteins of R. flavefaciens 17. In addition, cohesins of ScaA and ScaB skeletal proteins of R. flavefaciens B34b can be mentioned. Moreover, the cohesin in each skeleton protein of ScaA and ScaB of R. flavefaciens C94T is mentioned. Further, cohesins in the ScaA, ScaB, ScaC, and ScaE skeletal proteins of R. flavefaciens FD-1. Among them, cohesin in the ScaB skeletal protein of R. flavefaciens 17 or R. flavefaciens FD-1 can be mentioned. In the scaffold protein, cohesins are arranged in one to plural pieces with an appropriate linker sequence. One or a combination of two or more regions containing at least a part of the scaffold protein and containing one or more cohesins can be used.

  The amino acid sequences of the ScaA, ScaB, ScaC, and ScaE backbone proteins of R. flavefaciens 17 are represented by SEQ ID NOs: 1, 2, 3, and 4. The amino acid sequences of the ScaA and ScaB skeletal proteins of R. flavefaciens B34b are represented by SEQ ID NOs: 5 and 6, respectively. Furthermore, the amino acid sequences of the ScaA and ScaB skeletal proteins of R. flavefaciens C94T are represented by SEQ ID NOs: 7 and 8, respectively. Furthermore, the amino acid sequences of the ScaA, ScaB, ScaC, and ScaE skeletal proteins of R. flavefaciens FD-1 are represented by SEQ ID NOs: 9-12.

  The type III cohesin may be any cohesin obtained from various natural R. flavefaciens strains and classified as type III. Further, the type III cohesin only needs to have a binding activity to type III dockerin, and may be a natural type III cohesin appropriately modified. The type III dockerin that binds to type III cohesins has been determined from R. flavefaciens. Examples of such type III dockerins include Cel44A dockerin (amino acid sequence; SEQ ID NO: 14, nucleotide sequence; SEQ ID NO: 13), ScaA dockerin (amino acid sequence; SEQ ID NO: 16, nucleotide sequence; SEQ ID NO: 15), ScaB-X dockerin ( Amino acid sequence; SEQ ID NO: 18, base sequence; SEQ ID NO: 17). These correspond to cohesins on the R. flavefaciens skeletal proteins already described.

  Examples of such a natural type III cohesin variant include a variant intended to suppress sugar chain modification at one or more N-type sugar chain modification sites that may be possessed by type III cohesin. In addition, in the amino acid sequence represented by NX-T (amino acid other than asparagine-proline-threonine) or NX-S (amino acid other than asparagine-proline), the N-type sugar chain modification expected site N is known to be located at N (A. Herscovics et al., The FASEB Journal (6): 540-550 (1993)). The site for N-type sugar chain modification in dockrin can be detected by appropriately using a database or the like.

  As a variant for suppressing the sugar chain modification of one or more N-type sugar chain modification sites in type III cohesin, the amino acid residue other than asparagine in N (asparagine) of the N-type sugar chain modification site The modification which has substitution of these is mentioned. Examples of other amino acid residues include phenylalanine, arginine, glutamic acid, glycine, leucine, serine, histidine, threonine, valine, alanine, lysine, proline, tryptophan, cysteine, aspartic acid, tyrosine, serine, methionine, and the like. .

  In addition, as other variants for suppressing sugar chain modification, amino acid residues other than serine and threonine of serine or threonine downstream of asparagine two residues at one or more N-type sugar chain modification sites It may be a variant having a substitution. Examples of other amino acid residues include phenylalanine, arginine, glutamic acid, glycine, leucine, histidine, valine, alanine, lysine, proline, tryptophan, cysteine, aspartic acid, tyrosine, serine, methionine, and asparagine.

  In order to introduce an amino acid residue other than asparagine into N of the N-type sugar chain-modified site, or to introduce other amino acid residues into S / T at the same site, Site-directed mutagenesis techniques can be used.

  As a variant related to the N-type sugar chain modification site, one or more N-type sugar chain modification sites N and / or S / T on type III cohesin are each substituted with another amino acid residue. As long as it suffices, N and / or S / T of all N-type glycosylation sites on type III cohesin may be substituted with other amino acid residues, respectively.

  Still another variant is a variant having a continuous deletion of 100 residues or less, preferably 90 residues or less, more preferably 80 residues or less of the N terminus of type III cohesin. Can be mentioned.

  Moreover, it is preferable that the natural type III cohesin variant has 80% or more identity with the amino acid sequence of the natural type III cohesin. More preferably, it is 85% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more, and even more. Preferably it is 98% or more, More preferably, it is 99% or more.

  In the present specification, the identity and similarity in the amino acid sequence and the base sequence are determined by comparing the sequences, as known in the art, two or more proteins or two or more polynucleotides The relationship between. In the art, “identity” refers to a protein or polynucleotide sequence 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 between. 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 such 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). Similarity is referred to as Similarity in the BLAST sequence homology search results described below. Preferred methods for determining identity and similarity are designed to align the longest between the sequences being compared. The method for determining identity and similarity is coded in a publicly available program. 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.

Whether or not the modified type III cohesin has binding activity with the type III dockin is determined by, for example, displaying a test skeletal protein having one or more modified type III cohesins on the cell surface on a eukaryotic microorganism such as yeast. The transformant thus made can be evaluated by contacting with the type III dockin-carrying protein and detecting the amount of the type III dockerin-carrying protein held on the test scaffold protein. For the evaluation of the amount of the type III dockin-carrying protein, the type III dockerin-carrying protein may be a fluorescent protein that can be fluorescently discriminated, or the type III dockerin-carrying protein may be secondarily labeled with a fluorescent protein. Alternatively, the type III dockerin-containing protein itself may be a protein having an evaluable activity, or a protein having a secondary evaluable activity may be bound to evaluate the activity of the protein (enzyme). Good.
The specific activity of the protein may be measured. For example, when the target protein has a cellulase active site, the eukaryotic microorganism surface-displayed with the test skeletal protein is contacted with a type III dockerin-containing protein having a cellulose active site, and then separated eukaryotic. Enzyme activity can be evaluated by reacting a microorganism with an appropriate cellulase substrate (carboxymethylcellulose, cellulose phosphate, crystalline cellulose, etc.) and measuring the amount of reaction product and the base mass. Methods for quantifying the amount of reducing sugar produced as a result of such cellulase reactions include the Somogyi method, the Tauber-Kleiner method, the Hanes method (titration method), the Park-Johnson method, the 3,5-dinitrosalicylic acid (DNS) method, and the TZ method (Journal of Biochemical Methods, 11 (1985) 109-115) and the like may be appropriately employed.

  The skeletal protein of this artificial skeletal material has one or more of these type III cohesins. Preferably, 20 or less are provided. According to the present inventors, more than 20 type III cohesins have little effect on the enhancement of the function of the target protein.

  The scaffold protein preferably comprises two or more type III cohesins. Preferably it is in tandem. “Preparing in tandem” refers to a state in which two or more cohesins are arranged with an appropriate intervening sequence on the amino acid sequence (primary structure) of the backbone protein.

  The type III cohesin in the backbone protein is preferably arranged via an intervening sequence (linker) having an appropriate length in consideration of the accumulation degree of the target protein and the like. The intervening sequence between cohesins is not particularly limited, but one or two or more intervening sequences intervened between a plurality of cohesins in an ScaB protein derived from R. flavefaciens that retains a natural type III cohesin is appropriately used. Can be provided. The amino acid sequence between cohesins in this skeletal protein is determined by linking the necessary number of amino acids of several to 20 or less originally present in the upstream and downstream of the cohesin III in the skeletal protein from which cohesin III is obtained. can do.

  The scaffold protein preferably comprises three or more type III cohesins. This is because when the number is less than 3, the accumulation effect and proximity effect of the target protein are hardly exhibited. More preferably, four or more type III cohesins are provided. This is because when the number is 4 or more, the function enhancement of the target protein becomes clear due to the accumulation / proximity effect of the target protein. More preferably, it is 6 or more, still more preferably 8 or more, and still more preferably 10 or more. More preferably, it is 11 or more, and most preferably about 12. The upper limit is preferably 18 or less, more preferably 16 or less, and still more preferably 14 or less. More preferably, it is 13 or less.

  When the backbone protein comprises a relatively large number of type III cohesins, for example, 5 or more, preferably 6 or more, more preferably 8 or more, more preferably 10 or more, particularly preferably 12 type III. When comprising cohesin, all the N-type sugar chain modification scheduled sites on the backbone protein may not be modified in such a manner that sugar chain modification is suppressed. Due to the glycosylation of proteins by eukaryotic microorganisms, extracellular secretion (including surface presentation) of this skeletal protein with more type III cohesins, ie with a longer amino acid sequence, is completely avoided. This is because it tends to be suppressed. Therefore, the total number of type III cohesins, the total number of N-type glycosylation sites to be modified, and the number of variants to the non-glycosylated mode are appropriately adjusted on the backbone protein holding such a number of type III cohesins. Thus, it is important to optimize the number of type III cohesins for binding the target protein and the amount of sugar chain binding. By such adjustment, it is possible to secure the effect of accumulation of the target protein by the present backbone protein that is finally presented or secreted outside the cell.

  On the other hand, when the backbone protein comprises 4 or less type III cohesins, it is preferable that a plurality of N-type sugar chain modification sites that can be included in these cohesins are generally substituted in an unmodified modification mode, , 60% or more, more preferably 65% or more, more preferably 70% or more, still more preferably 75% or more, more preferably 80% or more, even more preferably, of the N-type glycosylation site to be included in type III cohesin Preferably, 85% or more, more preferably 90% or more, and more preferably 95% or more of the N-type sugar chain modification sites are altered to an unmodified form.

  When the backbone protein comprises two or more cohesins, the cohesin may comprise a plurality of single cohesins having the same amino acid sequence, or a combination of two or more cohesins having different amino acid sequences. Also good. When it is intended to provide a heterologous target protein in an intended sequence on the backbone protein, a plurality of types of cohesins having different binding specificities may be intentionally arranged. In this case, the target protein is also provided with a dockin that exhibits a cohesin-docklin interaction according to the binding specificity of cohesin.

  The backbone protein may further comprise a cohesin other than the cohesin having the binding activity to the above-described type III dockerin. For example, the above-mentioned type I cohesin and type II cohesin can be mentioned.

  For example, other cohesins include C. thermocellum CipA-derived cohesins. Many of the amino acid sequences and DNA sequences of other cohesins in the cellulosome of various cellulosome-producing microorganisms have been determined. The amino acid sequence and DNA sequence of these various CBDs can be easily obtained from various protein databases and DNA sequence databases accessible via NCBI website (http://www.ncbi.nlm.nih.gov/). Can be obtained.

  In addition to these various cohesins, the backbone protein preferably has a cellulose-binding domain (CBD) of a backbone protein selected from types I to III. CBD is known as a domain that binds to cellulose, which is a substrate in various skeletal proteins (the above-mentioned Kaburaku et al.). The cellulose binding domain may have one or two or more. Many of the amino acid sequences and DNA sequences of CBD in the cellulosome of various cellulosome-producing microorganisms have been determined. As with other cohesins, the amino acid sequences and DNA sequences of these various CBDs can also be easily obtained from various accessible protein databases and DNA sequence databases.

  The backbone material only needs to contain the backbone protein, and may contain the backbone protein in any form. For example, the backbone protein may be included in a free state, that is, without supporting the backbone protein with any carrier. In addition, the backbone material may be held on the surface of a carrier having a surface constructed so that the backbone protein can be appropriately held, such as a suitable carrier, for example, a column filler or a bead. If necessary, the scaffold protein is appropriately provided with a supply-binding functional group and a non-covalent binding region so as to be capable of binding to such a carrier.

  Furthermore, as will be described later, the scaffold protein may be in a state presented on the cell surface of a eukaryotic microorganism. In the form presented on the cell surface layer, the skeletal protein itself can be easily accumulated at a site called the cell surface layer easily by producing and presenting the skeletal protein in a eukaryotic microorganism. In order to present the present skeletal protein on the cell surface layer of the eukaryotic microorganism, the skeletal protein may be supplied from the outside to the cell surface layer of the eukaryotic microorganism and held.

  In order to impart cell surface display properties to this skeletal protein, known secretion signals and surface display systems can be used. For example, a secretory signal, an aggregating protein, or a part of the amino acid sequence is given. Examples of the secretion signal include secretion signals of glucoamylase genes of Rhizopus oryzae and C. albicans, yeast invertase leader, α factor leader and the like. Examples of the aggregating protein include a peptide consisting of 320 amino acid residues in the 5 'region of the SAG1 gene encoding α-agglutinin. Polypeptides and techniques for displaying a desired protein on the cell surface are disclosed in WO01 / 79483, JP2003-235579, WO2002 / 042483, WO2003 / 016525, and JP2006. No. 136223, 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.

(DNA encoding the backbone protein)
The DNA encoding the backbone protein may be any DNA as long as it encodes the amino acid sequence of the backbone protein. However, the DNA base sequence is determined according to the codon usage in eukaryotes to be degenerated or expressed in the genetic code. Any one may be used as long as it encodes a predetermined amino acid sequence without changing the amino acid sequence. For example, when expression in yeast is intended, a known yeast optimized codon may be applied.

  Such DNA may encode the amino acid sequence of type III cohesin and intervening sequences and other amino acid sequences of other desired elements based on the design of the backbone protein. Various coding DNAs can be obtained from a known database in the same manner as the amino acid sequence of type III cohesin. Such DNA can be obtained by a known technique in terms of genetic engineering or chemical synthesis.

(Production of the backbone protein)
This skeletal protein includes, for example, a base sequence encoding cohesin, a base sequence encoding an amino acid sequence of a moderately long intervening sequence, a base sequence encoding CBD, a secretory signal, and a surface layer display as necessary. A DNA encoding the skeletal protein is obtained by combining a base sequence combined with a base sequence encoding an amino acid sequence for use, and a recombinant vector or the like is constructed by a known method. The construction of such a recombinant vector is well known to those skilled in the art, and a person skilled in the art can construct a desired vector by appropriately selecting various necessary elements other than the coding DNA and the construction method thereof. A suitable host such as a eukaryotic microorganism such as yeast is transformed with the recombinant vector, the transformed cell is cultured according to a conventional method known to those skilled in the art, and the backbone protein is recovered from the cultured cell or medium. Can be obtained. In addition, as described above, the backbone protein may be obtained so as to be secreted or displayed on the cell surface of the eukaryotic microorganism. This skeletal protein is excellent in productivity and secretion (cell surface presentation) when expressed in a eukaryotic microorganism.

  This artificial skeletal material is preferable for placing or holding the target protein in an accumulated / close proximity. By doing so, the function of the target protein, for example, an enzyme activity can be improved if it is an enzyme, and in the case of two or more target proteins, the cooperative action can be further improved. Further, since the target protein can be accumulated / closed and held or held, it can be used as a carrier for efficient separation, recovery and aggregation of proteins. The artificial skeletal material can also be used as a carrier for accumulating and holding the target protein. The target protein will be described in detail later.

  In addition, as described above, the present skeletal protein is suitable for production and extracellular secretion (including cell surface display) in eukaryotic microorganisms as compared with other type I and II skeletal proteins. Even higher integration is possible.

(Protein composite materials)
The protein composite material disclosed in the present specification includes the artificial skeletal material disclosed in the present specification and a dockrin derived from a type III dockerin and having a binding activity to a type III cohesin (hereinafter simply referred to as a type III dockern). One or more target proteins each having According to the protein composite material disclosed in the present specification, the target protein can be accumulated / closed and held. This makes it possible to obtain a complex in which the target protein is highly integrated and the function of the target protein is improved and the cooperative action of two or more target proteins is improved.

  In the present specification, the target protein is not particularly limited in its function as a protein. Preferably, it is a protein whose function is improved by integration or proximity. Examples of such proteins include proteins having enzyme activity. Examples of such proteins include proteins having cellulase activity. A known cellulase can be appropriately used as the cellulase. Cellulases include endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 23.2.4.1, EC 3.2.1.21). Note that cellulase 13 (5, 6, 7, 8, 9) of GHF (Glycoside Hydrolase family) (http://www.cazy.org/fam/acc.gh.html) is based on the similarity of amino acid sequences. , 10, 12, 44, 45, 48, 51, 61, 74). As cellulases, the same or different cellulases classified into different families may be combined.

  In consideration of utilization of saccharification of biomass containing cellulose, the cellulase preferably contains two or more selected from the group consisting of β-glucosidase, endoglucanase and cellobiohydrolase.

  The cellulase is not particularly limited, but is preferably a cellulase having high activity by itself. Examples of such cellulases include, for example, Phanerochaete bacteria such as Phanerochaete chrysosporium, Trichoderma bacteria such as Trichoderma reesei, Fusarium bacteria, Tremetes bacteria, Penicillium bacteria In addition to fungi, Humicola, Acremonium, Aspergillus, and other filamentous fungi, Clostridium thermocellum, Clostridium cellulolyticum and other Clostridium, Pseudomonas Bacteria, bacteria such as Cellulomonas, Ruminococcus, Bacillus, protozoa such as Sulfolobus, and Streptomyces, thermoactino Derived from radioactive bacteria such as Myrces (Thermoactinomyces) A cellulase is mentioned. Such cellulase or its active site may be artificially modified.

  As such a cellulase, for example, PcCBH2 derived from Phanerochaete chrysosporium (SEQ ID NO: 19 (SEQ ID NO: 20); this notation with respect to the SEQ ID NO means the SEQ ID NO of the nucleotide sequence (SEQ ID NO of the amino acid sequence). And variants thereof. Further, an endoglucanase derived from Trichoderma reesei (SEQ ID NO: 21 (SEQ ID NO: 22)) and a modified form thereof can be mentioned. Further, cellulases derived from Clostridium thermocellum (SEQ ID NO: 23 (SEQ ID NO: 24), SEQ ID NO: 25 (SEQ ID NO: 26), SEQ ID NO: 27 (SEQ ID NO: 28)) and modified forms thereof may be mentioned.

  Moreover, when the effective utilization of biomass is taken into consideration, a hemicellulase active site may be provided. Further examples include lignin degrading enzymes such as lignin peroxidase, manganese peroxidase and laccase. In addition, examples include cellulose binding domains (proteins) that are constituent parts of cellulose relieving proteins swollenin and expansin, cellulosome and cellulase. In addition, other biomass degrading enzymes such as xylanase and hemicellulase can also be mentioned. Any of these proteins can improve the accessibility of cellulase to cellulose.

  The target protein is bound on the backbone protein by type III cohesin-dockerin interaction. For example, various type III dockins on the cellulosome of R. flavefaciens can be used according to the type III cohesin used on the backbone protein (ScaA, ScaB, ScaC and ScaE). When the scaffold protein uses ScaB cohesin as a type III cohesin, a ScaA dockerin can be used as a type III dockerin.

  The type III dockerin may be a natural type III dockerin or a variant that does not lose the binding property to the type III cohesin, similar to the above-described cohesin. For example, it may be a variant in which N (asparagine) at the site of N-type sugar chain modification on the type III dockerin is replaced with an amino acid residue other than asparagine. Further, it may be a modified form in which an S / T at the site of N-type sugar chain modification is substituted with an amino acid residue other than these. In such a modified body, sugar chain modification can be avoided and the original cohesin-dockerin binding strength can be secured. In these variants, as with type III cohesins, N and S / T may be widely substituted with other amino acid residues.

  Further, it is preferable that such a natural type III docker variant has 80% or more identity with the amino acid sequence of natural type III dockerin. More preferably, it is 85% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more, and even more. Preferably it is 98% or more, More preferably, it is 99% or more.

  The target protein has a type III dockerin in addition to its functional active site. The position of type III dockerin in the target protein is not particularly limited. It may be on the N-terminal side or on the C-terminal side. Both of these may have type III dockerins. Depending on the target protein, the preferred type III dockin position may differ. That is, the binding ability to type III cohesin or the activity of the target protein may differ depending on the position of type III dockin. For example, in the case of cellobiohydrolase PcCBH2 derived from Phanerochaete chrysosporium or endoglucanase of Trichoderma reesei, the type III dockerin is preferably located on the N-terminal side which may contain the N-terminal. In the case of cellulase derived from Clostridium thermocellum, the type III dockerin is preferably located on the C-terminal side which may contain the C-terminal.

(Production of target protein)
Such a target protein can be obtained by genetic engineering as a fusion protein in which a type III dockerin is fused to a desired target protein. That is, a recombinant vector is constructed based on a known technique using DNA encoding such a fusion protein, a suitable host of a eukaryotic microorganism such as yeast or a bacterium such as E. coli is transformed, and a transformed cell Can be obtained by culturing according to a conventional method known to those skilled in the art and recovering the backbone protein from the cultured cells or medium. Further, the target protein may be obtained so as to be secreted outside the cells of the eukaryotic microorganism using a known system as described above.

(Production of this composite material)
This composite material can be obtained by bringing the backbone protein and the target protein into contact with each other and binding them based on the cohesin-docklin interaction between the type III cohesin on the backbone protein and the type III dockerin of the target protein.

  The composite material can itself be present independently of cells such as eukaryotic microorganisms. In this case, for example, the present composite material based on the cohesin-docklin interaction can be obtained by bringing a free backbone protein that is not displayed on the cell surface into contact with a free target protein under conditions that can generally maintain protein folding. Is built. The contact condition may be mixing the two in a liquid having a pH, a salt concentration, and a temperature. The contact probability may be improved by stirring as appropriate.

  Such a composite material may be constructed by contacting the target protein with the backbone protein in a state separated from eukaryotic microorganisms or the like that produce each of the composite materials, or eukaryotes constructed so that each protein is secreted extracellularly. You may make it construct | assemble this composite material out of the cell of a eukaryotic microorganism by carrying out mixed culture of microorganisms etc. In such a case, for example, cellulase is used as the target protein, and eukaryotic microorganisms such as yeast are cultured in the presence of a cellulose-containing material, whereby cellulose saccharification and yeast fermentation can be simultaneously performed.

  Moreover, this composite material can also be provided in the cell surface layer of a eukaryotic microorganism. In this case, for example, by culturing eukaryotic microorganisms such as yeast in the presence of cellulose-containing material with the target protein as cellulase, saccharification of cellulose on the yeast cell surface layer and fermentation by yeast can be performed simultaneously. There are several ways to produce such a composite material. One is a method of supplying a target protein from the outside to a eukaryotic microorganism constructed so that the backbone protein is presented on the cell surface using a known cell surface display system. The backbone protein and the target protein construct the composite material on the cell surface of a eukaryotic microorganism based on the cohesin-docklin interaction.

  Further, in a eukaryotic microorganism constructed so that the present skeletal protein is presented on the cell surface using a known cell surface presentation system, the protein of interest is constructed to be extracellularly secreted using a known method. By culturing the eukaryotic microorganism constructed in such co-expression, the composite material can be constructed on the cell surface of the eukaryotic microorganism.

(Eukaryotic microorganism)
The eukaryotic microorganism disclosed in the present specification is a eukaryotic microorganism provided with the backbone protein on the cell surface layer. The eukaryotic microorganism can be provided with the present skeletal protein by a known cell surface display system. Such eukaryotic microorganisms preferably self-produce the backbone protein in cells. This is because this scaffold protein is suitable for production and secretion in eukaryotic microorganisms, and also simplifies the surface layer presentation and can efficiently and stably present the backbone protein on the surface layer.

  These eukaryotic microorganisms hold the DNA encoding the backbone protein in an expressible manner. Such DNA is only required to be capable of expressing the protein in a eukaryotic microorganism, and the retention form is not particularly limited. For example, it is linked under the control of a promoter operable in the host microorganism and is held in a state having an appropriate terminator downstream thereof. The promoter may be a constitutive promoter or an inducible promoter. The DNA in such a state may be in a form incorporated in the host chromosome, or in a form such as a 2μ plasmid retained in the host nucleus or a plasmid retained outside the nucleus.

  The eukaryotic microorganism disclosed in the present specification may also be a eukaryotic microorganism provided with the composite material comprising the backbone protein and the target protein on the cell surface layer. Such eukaryotic microorganisms can be provided with this backbone protein by a known cell surface display system. The target protein may be self-produced by co-expression or may be supplied from the outside. The self-production of the backbone protein and the target protein simplifies the surface layer presentation, and the surface layer protein can be efficiently and stably displayed on the surface layer.

  Such a eukaryotic microorganism can hold at least a DNA encoding the present backbone protein so that it can be expressed, and can also hold a DNA encoding the target protein so that it can be expressed. Such DNA is only required to be capable of expressing the protein in a eukaryotic microorganism, and the retention form is not particularly limited. For example, it is linked under the control of a promoter operable in the host microorganism and is held in a state having an appropriate terminator downstream thereof. The promoter may be a constitutive promoter or an inducible promoter. The DNA in such a state may be in a form incorporated in the host chromosome, or in a form such as a 2μ plasmid retained in the host nucleus or a plasmid retained outside the nucleus.

  In the surface layer of a eukaryotic microorganism, the present skeletal protein is not limited to be retained based on the already-described known cell surface display technology, and may be retained in another known form.

  In the eukaryotic microorganism disclosed in the present specification, the target protein preferably contains one or more cellulases as described above. By using such a eukaryotic microorganism, simultaneous saccharification and fermentation of cellulose can be performed efficiently. This is because the backbone protein can accumulate and hold one or more cellulases, which are target proteins, based on the type III cohesin-docklin interaction, and thus can achieve a high synergistic effect of these cellulases.

  In many cases, enzymes such as cellulase inherently have a signal for extracellular secretion. A known secretion signal can be used to impart extracellular secretion to a target protein such as cellulase. As described above, the secretion signal is appropriately selected according to the type of eukaryotic microorganism to be used.

  The eukaryotic microorganism is not particularly limited, and for example, various known yeasts can be used. In consideration of ethanol fermentation described later, yeasts of the genus Saccharomyces cerevisiae such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe, Candida shehatae, etc. Yeast of the genus Candida, yeast of the genus Pichia such as Pichia stipitis, yeast of the genus Hansenula, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, the genus Pachysolen And yeast of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis. Of these, Saccharomyces yeasts are preferable from the viewpoint of industrial availability. Of these, Saccharomyces cerevisiae is preferable.

(Production of transformed eukaryotic microorganisms, etc.)
The described eukaryotic microorganisms, the present skeletal protein, the target protein, etc. disclosed in the present specification are all methods described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, etc. It can produce according to. Vectors for transformation of eukaryotic microorganisms and methods for their construction are well known to those skilled in the art and are disclosed in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like. In addition, the form of a vector can take various forms according to a usage form. For example, it can take the form of a DNA fragment, and can also take the form of a suitable yeast vector such as a 2 microplasmid. A person skilled in the art can also perform transformation with a vector by referring to the above-mentioned book. Typically, various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and the like can be used.

(Recombinant vectors, etc.)
According to the present specification, in addition to an expression vector (recombinant vector) that holds the DNA encoding the backbone protein so that it can be expressed in a eukaryotic microorganism, the DNA encoding the target protein is expressed in a eukaryotic microorganism. An expression vector (recombinant vector) that can be retained is provided. Moreover, the vector set which made these a set is also provided. These vectors may be in the form of expression cassettes or in the form of DNA constructs capable of homologous recombination. Such a coding DNA may be incorporated into a commercially available vector. As already explained, the construction and form of such vectors are well known to those skilled in the art.

(Use of artificial skeletal materials and protein composite materials)
According to this specification, utilization of this artificial skeletal material and utilization of protein composite material are also provided. The artificial skeletal material can retain the target protein by, for example, contacting with the target protein, and therefore can be used for collection, separation, purification, etc. in addition to accumulation of the target protein. Moreover, the protein composite material holds the target protein by the cohesin-docklin interaction, and can contribute to the enhancement of the function and the cooperation of the target protein. Below, the form using the protein which has cellulase activity as a target protein is demonstrated.

(Method for producing saccharified product from cellulose)
According to the present specification, a method for producing a saccharified product such as glucose from a cellulose-containing material using the protein composite material disclosed in the present specification is provided. This method includes a step of bringing a protein composite material and a cellulose-containing material into contact with each other. The saccharified product is not limited to glucose, and various monosaccharides and disaccharides may be included depending on the composition of the cellulose-containing material and the type of cellulase used.

  In this method, the protein composite material holds at least one or more cellulases as target proteins. One or more cellulases may be combined with various cellulases as necessary. Since the cellulase in the protein composite material is integrated / proximal, the cellulase can be effectively decomposed to decompose cellulose. The contact condition between the cellulose-containing material and the protein composite material is not particularly limited as long as the cellulase functions. For example, it is preferable to employ a relatively low temperature condition of about 15 ° C. to about 25 ° C. At such a low temperature, it is considered that the protein composite material is in a folded state in which the backbone protein and cellulase are likely to act. In particular, when mold-derived cellulase is used, such low-temperature culture is preferably applied. Preferably it is about 18 degreeC or more and 22 degrees C or less, More preferably, it can be set as about 20 degreeC.

  When a eukaryotic microorganism such as yeast that displays the protein composite material on the surface is used as the protein composite material, a step of culturing or fermentation can be taken. Furthermore, yeast can utilize the decomposition product of cellulose to allow growth and fermentation. The culture conditions are not particularly limited as long as they are generally employed in eukaryotic microorganisms. Even in this case, a relatively low culture temperature of 15 ° C. or more and 25 ° C. or less can be employed as described above.

  Any cellulose-containing material may be used as long as it contains cellulose that is β-glucan in which D-glucose is glycosidically bonded with β-1,4 bonds. The cellulose-containing material may have any origin or form. Accordingly, examples of the cellulosic material include various cellulosic materials such as lignocellulosic material, crystalline cellulose material, soluble cellulose material (amorphous cellulose material), and insoluble cellulose material. Examples of the lignocellulosic material include lignocellulosic materials in a state in which lignin and the like are combined in the woody part and leaf part of a woody plant and the leaves, stems, roots and the like of a herbaceous plant. Examples of such lignocellulosic materials include rice straw, wheat straw, corn stover, bagasse and other agricultural waste, collected trees, branches, dead leaves, etc. or chips obtained by defibrating these, sawdust, chips, etc. It may be a waste from a sawmill factory, a forest residue such as thinned wood or damaged trees, and a construction waste. Examples of the crystalline cellulose material and the insoluble cellulose material include crystalline or insoluble cellulose materials containing crystalline cellulose and insoluble cellulose after separating lignin and the like from the lignocellulose material. As the cellulose material, used paper products such as used paper containers, used paper, used clothes, and pulp waste liquid may be used.

  The protein composite material can hold hemicellulase as a target protein together with cellulase. The protein composite material can also retain only hemicellulase as the target protein. Cellulose-containing materials often contain hemicellulose at the same time. These saccharified products (such as glucose and xylose) can also be produced by bringing such protein composite material into contact with a cellulose-containing material containing hemicellulose or a material mainly composed of hemicellulose.

(Production method of useful substances from cellulose)
The production method of a useful substance disclosed in the present specification includes a step of culturing a eukaryotic microorganism that assimilate a cellulose saccharified product in the presence of the protein composite material and the cellulose-containing material disclosed in the present specification. it can. According to this method, a saccharified cellulose can be effectively obtained from the protein composite material, and the eukaryotic microorganism can be proliferated and fermented therefrom. Therefore, the eukaryotic microorganism can simultaneously produce useful substances. In this method, preferably, a eukaryotic microorganism having a protein composite material on the cell surface layer is used. By carrying out like this, a useful substance can be more effectively produced from a cellulose content material.

  The eukaryotic microorganism is not particularly limited, but yeast is preferable because a culture method is established industrially and genetic engineering modification is easy.

  A useful substance is a product obtained by fermenting a nutrient source such as glucose by a eukaryotic microorganism, and varies depending on the type of eukaryotic microorganism and also on the fermentation conditions. Although it does not specifically limit as a useful substance, What is necessary is just what yeast and other eukaryotic microorganisms can produce using glucose. Useful substances are non-original metabolites that can be synthesized by replacing or adding one or more enzymes in the metabolic system from glucose in eukaryotic microorganisms such as yeast. May be. Useful substances include, for example, ethanol, etc., C3-C5 lower alcohols, organic acids such as lactic acid, fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.), glycolysis modification by addition of isoprenodo synthesis pathway Materials for biorefinery technology, such as glycerin produced from plastics and raw materials for plastics and chemical products.

  What is necessary is just to implement a culture | cultivation process according to the kind of cellulose-containing material to be utilized, a eukaryotic microorganism, and the useful substance to obtain. In addition, stationary culture, shaking culture, aeration stirring culture, or the like can be used for the culture. The aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. The culture temperature is not particularly limited, but may 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 about 6 to 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. In addition, after completion | finish of a conversion process, you may implement the process of removing microorganisms from a culture solution, collect | recovering fractions containing useful substances, such as ethanol, and also concentrating this.

  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, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination operation described below was performed according to Molecular Cloning 3rd edition.

(Acquisition of Ruminococcus flavefaciens-derived gene and production of yeast expression vector)
ScaA cohesin derived from luminococcus flavfaciens (SEQ ID NO: 29 (SEQ ID NO: 30)), Cel44A dockin (SEQ ID NO: 31 (SEQ ID NO: 32)), ScaB cohesin (SEQ ID NO: 33 (SEQ ID NO: 34)), ScaA dockin (SEQ ID NO: 35) (SEQ ID NO: 36)), ScaE cohesin (SEQ ID NO: 37 (SEQ ID NO: 38)) and ScaB-X Docklin (SEQ ID NO: 39 (SEQ ID NO: 40)), and the above six amino acid sequences were obtained from the UniProt database. A synthetic gene optimized for codon usage was prepared.

  The restriction enzyme sites of BglII and XhoI were added upstream of each of the synthesized ScaA, ScaB, and ScaE cohesin genes by PCR. Using this restriction enzyme site, as shown in FIG. 1, upstream ADH3 homology region (ADH3U), HOR7 promoter and Rhizopus orizae glucoamylase-derived signal sequence (described as SSRG: SEQ ID NO: 41), downstream V5 tag, PDL-HOR7p-ScaAcoh-AGA2, pDL-HOR7p-ScaBcoh-AGA2, and pDL-HOR7p-ScaEcoh-AGA2 vectors having an AGA2 sequence, TDH3 terminator, Leu3 marker and ADH3 homology region (ADH3D) were prepared.

  Similarly, the restriction enzyme sites of XhoI and BamHI were added upstream of each of the Cel44A dockin, ScaA dockin, and ScaB-X dockerin genes synthesized by PCR. Using this restriction enzyme site, as shown in Fig. 2, the HXT3 homology region (HXT3U), HOR7 promoter, SSRG signal sequence and His-tag are upstream, and the TDH3 terminator, Ura3 marker and HXT3 homology region (HXT3D) are downstream. PHU-HOR7p-Cel44Adoc, pHU-HOR7p-ScaAdoc, and pHU-HOR7p-ScaB-Xdoc were prepared.

(Production of various genes on the surface of yeast)
As shown in FIG. 3, pAI-HOR7p-, which has an AAP1 homology region (AAP1U) and HOR7 promoter upstream of the AGA1 gene cloned by PCR and has a TDH3 terminator, His3 marker and AAP1 homology region (AAP1D) downstream. AGA1 vector was prepared. This vector was linearized with the restriction enzyme Sse8387I, transformed into the yeast S. cerevisiae BY4741 strain, and homologous recombination was performed to obtain yeast BYAGA1 presenting AGA1 on the cell surface. Various vectors pDL-HOR7p-ScaAcoh-AGA2, pDL-HOR7p-ScaBcoh-AGA2, pDL-HOR7p-ScaEcoh-AGA2 prepared in Example 1 were introduced into BYAGA1 strain by linearizing with Sse8387I, BYScaA, BYScaB, BYScaE strain It was. In addition, pHU-HOR7p-Cel44Adoc, pHU-HOR7p-ScaAdoc, pHU-HOR7p-ScaB-Xdoc were linearized with Sse8387I and introduced into BY4741 strain to obtain BYCel44Adoc, BYScaAdoc, BYScaB-Xdoc strains.

(Evaluation of the amount of yeast surface displayed by flow cytometry (FCM))
The ScaB cohesin-ScaA dockerin combination has already been evaluated (detailed in JP 2011-160772). Therefore, the amount of ScaA, ScaE cohesin presented to the yeast surface layer was evaluated using the FCM for the amount of ScaA cohesin-Cel44A dockin and ScaE cohesin-ScaB-X dockin. Each cohesin-presenting yeast was cultured in YPD liquid medium (10 g / l yeast extract, 20 g / l peptone, 20 g / l glucose) at 30 ° C. for 24 hours, stained with AntiV5-FITC antibody, and then on the yeast cell surface with FCM. Cohesin display amount was evaluated. As a result, as shown in FIG. 4, the amount of ScaA cohesin presented was very small compared to ScaE cohesin.

  Next, by adding culture supernatants of BYCel44Adoc and BYScaB-Xdoc strains that secrete and produce the corresponding dockrin to these cohesin-displaying yeasts, minicellulosomes were reconstituted on the yeast surface layer. The dockin bound to cohesin was labeled with an AntiHis-FITC antibody, and the amount of dockin bound was evaluated by measuring with FCM. As a result, as shown in FIG. 5, the combination of ScaA cohesin and Cel44 dockerin showed a very small amount of dockin compared to the combination of ScaE cohesin and ScaB-X dockerin. From the above results, it was considered that the combination of ScaA cohesin and Cel44 dockerin was not suitable for expression in yeast.

  Combination of ScaB cohesin-ScaA dockin and ScaE cohesin-ScaB-X dockerin and clostridium thermocellum CipA cohesin and Ct48SDD (Cel48S dockin's two N-type sugar chain binding sites (N-type sugar chain modification sites) asparagine) Substitution with aspartic acid, described in JP-A-2011-219399)), the above three types of cross-linking properties were evaluated.

  Each cohesin-presenting yeast was cultured in YPD liquid medium at 30 ° C. for 24 hours, and the culture supernatant of each dockerin-secreting yeast was added in all combinations to reconstitute minicellulosomes on the yeast surface layer. Dockin bound to cohesin was labeled with AntiHis-FITC antibody, and the amount of binding was evaluated by FCM. As a result, as shown in FIG. 6, ScaB cohesin-ScaA dockin and ScaE cohesin-ScaB-X dockrin produced in yeast retain selective binding with other combinations, and the combination of ScaB cohesin-ScaA dockin is the most. There was a lot of dockin binding.

(Production of ScaB-4 cohesin (Rf4Coh) on the surface of yeast)
Example 3 shows that ScaB cohesin is suitable as a skeletal protein element in yeast. Therefore, a synthetic gene was prepared by optimizing R.flavefaciens-derived ScaB 4 cohesin for yeast codon use (SEQ ID NO: 42). ScaB-4 cohesin (Rf4Coh) display yeast was produced. Specifically, a restriction enzyme site of BglII was added upstream of Rf4Coh, and a restriction enzyme site of XhoI was added downstream by PCR. Utilizing this restriction enzyme site, pDL-Rf4coh with ADH3 homology region (ADH3U) upstream, HOR7 promoter and SSRG signal sequence, V5 tag, AGA2 sequence, TDH3 terminator, Leu3 marker and ADH3 homology region (ADH3D) downstream -AGA2 vector was prepared (Fig. 1).

  Similar to Example 2, the obtained vector was used as a hygromycin marker and introduced into yeast BJ5465. By homologous recombination with BJ-AGA1, the yeast displaying Rf4Coh on the surface, BJRf4Coh strain was prepared. This strain was cultured overnight in a YPD medium, labeled with an AntiV5-FITC antibody in the same manner as in Example 3, and flow cytometry was performed. As a result, as shown in FIG. 7, it was confirmed that Rf4Coh was produced on the yeast surface layer.

(ScaA dockrin (RfDoc) fusion PcCBH2 and TrEG2 gene construction)
A cellulase expression vector for fusion expression of R.flavefaciens-derived ScaA dockerin (RfDoc) at the N-terminus, C-terminus, and both ends was prepared as follows. Specifically, three types of cassettes (for N-terminal, C-terminal, and both-terminals) were amplified using PCR, and the In-Fusion Advantage PCR Cloning Kit (Takara Bio) was added to the SphI site of pRS436-GAPSSRG (Figure 8). ) (FIG. 9). Three types of ScaA dockin fusion secretion expression vectors can be fused to the N-terminal, C-terminal, and both ends by inserting any cellulase into the NheI site using the In-Fusion Advantage PCR Cloning Kit.

  Using this vector, Phanerochaete chrysosporium-derived cellobiohydrolase II (PcCBH2, SEQ ID NO: 43) and Trichoderma reesei-derived endoglucanase II (TrEG2, SEQ ID NO: 44) were fused with ScaA dockin at both ends. Produced.

(Evaluation of PSC degradation activity of PcCBH2, TrEG2 display yeast)
The constructed vector was transformed into the BJRf4Coh strain prepared in Example 4 using Frozen-EZ Yeast Transformation II Kit (Zymo Research). Inoculate the grown bacterial colonies in 500 μl of SD-URA liquid medium (yeast nitrogen base without amino acids without ammonium sulfate 1.7 g, casamino acid 10 g, -URA amino acid mix 0.77 g, glucose 10 g, deionized water 1000 ml) The bacterial solution cultured at 30 ° C. for 20 hours was used as a preculture solution. 100 μl of the preculture was newly inoculated into 500 μl of SD-URA liquid medium and cultured at 30 ° C. for 20 hours for main culture. The cells were collected by centrifugation, and subjected to a phosphate swelling cellulose (PSC) degradation test using the cells after washing with 50 mM citrate buffer (pH 5.0).

  100ul of cells suspended in 200ul of 50mM citrate buffer (pH 5.0) was added to 100ul of 1% PSC solution, and after reaction at 40 ° C for 20-24 hours, the amount of reducing sugar in the centrifugal supernatant was determined by TZ assay. It was measured by the method (Journal of Biochemical and Biophysical Methods, 11 (1985) 109-115). As shown in FIG. 10, all reactions were performed in triplicate. As a result, both PcCBH2 and TrEG2 showed the highest PSC degradation activity when ScaA dockin was fused to the N-terminus.

(Evaluation of PSC degradation activity of various cellulase presenting yeasts)
PcCBH2 mutant, ThCBD1-PcMal4 (L2) (SEQ ID NO: 45 (SEQ ID NO: 46)), TrEG2 mutant, ThCBD1-TrEG2 (L4) (SEQ ID NO: 47 (SEQ ID NO: 48)), Phanerochaete chrysosporium-derived endoglucanase II Using the method of Example 5, the ScaA dockerin was fused to the N-terminus of (PcEG2, SEQ ID NO: 49 (SEQ ID NO: 50)). In addition, the original dockrins of Clostridium thermocellum-derived cellulases Cel9K, Cel8A, and CbhA were deleted (DNA sequence after removal of dockrin: CtCel9K, SEQ ID NO: 51), (CtCel8A, SEQ ID NO: 52) (CtCbhA, sequence No. 53), and a gene in which ScaA dockin was fused to the N or C terminus was prepared. Using these genes, the PSC degradation activity was measured by the same method as in Example 6.

  As a result, as shown in FIG. 11, ThCBD1-PcMal4 (L2) showed the highest activity for cellobiohydrolase, and ThCBD1-TrEG2 (L4) showed the highest activity for endoglucanase. In addition, Clostridium thermocellum-derived cellulase was more active when fused to the C-terminus that originally had dockrin than to fuse ScaA dockrin to the N-terminus.

(Comparison of activity during low temperature culture)
The culture temperature was 20 ° C. or 30 ° C., PSC decomposition activity was measured in the same manner as in Example 7, and the influence of the culture temperature was examined. As a result, as shown in FIG. 12, PSC decomposition activity was improved by culturing at 20 ° C. In particular, significant improvement in PSC degradation activity was observed in the cellulase-derived strains derived from fungi. Since it is well known that protein folding is improved by culturing at low temperature, it was found that low-temperature culturing is effective for fungal cellulase fused with dockerin.

(Docklin modification using local mutation and random mutation)
As shown in FIG. 13, the ScaA dockrin has one N-type glycosylation sequence, and it is predicted that a sugar chain is added when expressed in yeast, resulting in steric hindrance due to binding to cohesin. It was done. Moreover, since the cell activity was improved by low-temperature culture in Example 8, the effect of improving the ScaA dockin folding was expected. Therefore, the N-type sugar chain of ScaA dockrin was removed by evolutionary engineering, and ScaA dockrin was modified by introducing random mutations throughout the ScaA dockin.

The flow of the experiment is shown in FIG. Specifically, it was performed as follows.
(1) An NNK (N = ATGC, K = GT) mutation was introduced into the asparagine of the N-type glycosylation sequence shown on the ScaA dockin sequence in FIG.
(2) A random mutation was introduced to the full length by GeneMorph II Random Mutagenesis kit (Agilent Technology) using a library containing NNK mutation as a template.
(3) Transform the cellulase (ThCBD1-Mal4 (L2)) gene and mutation library into BJRf4Coh using Frozen-EZ Yeast Transformation II Kit (Zymo Research), and use secreted expression vector by using homologous recombination in cells Subcloned into For secretory expression, pAUR112-GAPSSRG with the TDH3 promoter-SSRG-CYC terminator inserted into the SmaI site of pAUR112 (Takara Bio), which is a low-copy vector, to reduce errors due to vector copy number between clones (FIG. 15). Was used.
(4) The PSC degradation activity of the bacterial cells was measured and screened.

  PSC degradation activity was measured in the same procedure as in Example 6, and a total of 864 clones were screened for 9 96-well plates as the primary screening. Next, a library having combinatorial mutations of the top 16 clones was prepared, and secondary screening of a total of 192 clones for two 96-well plates was performed by the same procedure as in Example 6. The upper clones were also retested in triplicate. As a result, as shown in FIG. 16, the clone with the highest activity had a PSC degrading activity up to about 2.2 times that of the wild type ScaA dockerin, and the ScaB cohesin and ScaA dockerin were bound by mutagenesis. It was predicted that power improved. Moreover, the sequence analysis of the obtained upper clone was performed. The results are shown in FIG. Among these, F7 having the most improved PSC degradation activity was designated as RfDoc (mut) and used in the subsequent experiments. The amino acid sequences of C6, G6, B7, D7, E7 and F7 shown in FIG. 17 are shown in SEQ ID NOs: 82 to 87, respectively.

(Glycan removal from Rf4Coh by local mutagenesis)
Rf4Coh has 9 N-type glycosylation sequences, and it was predicted that sugar chains were added when expressed in yeast, and steric hindrance occurred due to binding to dockerin (FIG. 18). Therefore, the following experiment was conducted with the purpose of simultaneously removing nine sugar chains. The flow of the experiment is shown in FIG. Specifically, NNK mutations were introduced into all glycosylation sequences by preparing 8 fragments with NNK local mutagenesis for 9 glycosylation sequences and linking them by overlapping PCR. A mutant library was prepared. The mutation library was subcloned into pAUR112-GAPSSRG, which is a low copy vector, and transformed into BJ-AGA1 strain using Frozen-EZ Yeast Transformation II Kit (Zymo Research) (RfcohΔG library).
Next, RfDoc (mut) obtained in Example 9 was transferred to ThCBD1-Mal4 (L2) at the SphI site of pRS434-GAPSSRG (FIG. 20), a high-copy vector having a tryptophan marker and a TDH3 promoter-SSRG-CYC terminator. ) RfDoc (mut) -ThCBD1-Mal4 (L2) (SEQ ID NO: 54 (SEQ ID NO: 55)) fused to the N-terminus of the RfcohΔG library using Frozen-EZ Yeast Transformation II Kit (Zymo Research) A cellulase co-expressing RfcohΔG library was prepared by transforming to.

  Growing bacterial colonies in 500 μl of SD-URA-Trp liquid medium (yeast nitrogen base without amino acids without ammonium sulfate 1.7 g, casamino acid 10 g, -URA-Trp amino acid mix 0.72 g, glucose 10 g, deionized water 1000 ml) The bacterial solution which was inoculated and cultured at 30 ° C. for 20 hours was used as a preculture solution. 100 μl of the preculture was newly inoculated into 500 μl of SD-URA-Trp liquid medium and cultured at 30 ° C. for 20 hours for main culture. The cells were collected by centrifugation, and subjected to a phosphate swelling cellulose (PSC) degradation test using the cells after washing with 50 mM citrate buffer (pH 5.0).

  Add 100ul of cells suspended in 200ul of 50mM citrate buffer (pH 5.0) to 100ul of 1% PSC solution, and after reaction at 40 ° C for 20-24 hours, reduce the amount of reducing sugar in the supernatant by TZ assay Measured by the method. Screening was performed on a total of 192 clones for two 96-well plates. Next, the top 10 clones were selected and the same PSC degradation test was performed in triplicate. As a result, as shown in FIG. 21, the clone with the highest activity had an activity improvement of about 1.7 times compared to before the removal of the sugar chain. In addition, FIG. 22 shows the results of sequence analysis of the obtained upper 7 clones. As shown in FIG. The amino acid sequences of cohesins of 1, 2, 3, 7, 8, 9 and 10 are shown in SEQ ID NOs: 88 to 94, respectively.

(Examination of optimal length of ScaB cohesin derived from Luminococcus flavofaciens)
In order to determine the optimum length when ScaB cohesin derived from Luminococcus flavfaciens was presented to the surface of S. cerevisiae, ScaB cohesin-presenting yeasts of various lengths were prepared. Specifically, the pDL-Rf3cohAGA2 vector was prepared by deleting the C-terminal 1 cohesin of the pDL-Rf4cohAGA2 vector prepared in Example 4 by PCR. In addition, by introducing multiple units of the N-terminal 2 cohesin of ScaB-4 cohesin or 4 cohesin into the XhoI site of the pDL-Rf4cohAGA2 vector, pDL-Rf 6, 8, 12, 16, A 20 cohAGA2 vector was constructed. By homologous recombination of the obtained vector with BJ-AGA1, ScaB-3, 4, 6, 8, 12, 16, 20 cohesin (SEQ ID NO: 56 (SEQ ID NO: 57), SEQ ID NO: 58 (SEQ ID NO: 59), SEQ ID NO: 60 (SEQ ID NO: 61), SEQ ID NO: 62 (SEQ ID NO: 63), SEQ ID NO: 64 (SEQ ID NO: 65), SEQ ID NO: 66 (SEQ ID NO: 67), SEQ ID NO: 68 (SEQ ID NO: 69)) are presented on the surface layer. Yeast (multicohesin yeast) was prepared. The cells were cultured overnight in YPD medium, labeled with AntiV5-FITC antibody in the same manner as in Example 3, flow cytometry was performed, and the amount of cohesin presented on the surface of yeast was compared. As a result, as shown in FIG. 23, it was found that although the amount of presentation decreased with the cohesin length, it was also presented on the surface of the yeast cell even with a length of 20 cohesins.

(Evaluation of PSC degradation activity using multi-cohesin yeast (cellulase simultaneous expression))
An expression vector was prepared by subcloning RfDoc (mut) -ThCBD1-Mal4 (L2) obtained in Example 9 into the SphI site of pRS436-GAPSSRG (FIG. 8), and Frozen-EZ Yeast Transformation II Kit (Zymo Research) was prepared. Used to transform each multi-cohesin yeast. The PSC decomposition activity of the grown cells was measured in the same manner as in Example 6. The results are shown in FIG. In addition, 100 ul of the cell suspension was added to 100 μl of 2% Avicel solution, and after the reaction at 40 ° C. for 20 to 24 hours, the amount of reducing sugar in the centrifugal supernatant was measured by TZ assay method, and the Avicel degradation activity was also improved. It was measured. The results are shown in FIG. As shown in FIGS. 24 and 25, the cellulolytic activity was highest when both PSC and Avicel degrading activities were 12 cohesins.

(Evaluation of PSC degradation activity using multi-cohesin yeast (external addition of cellulase))
CtCel8A-RfDoc (mut) (SEQ ID NO: 70 (SEQ ID NO: 71)) and CtCbhA-RfDoc (mut) (SEQ ID NO: 72 (SEQ ID NO: 73)) obtained by fusing RfDoc (mut) obtained in Example 9 to the C-terminus Subcloning into the pET23b vector so as to be T7p-cellulase gene-His tag-T7t, an E. coli expression vector was prepared. The prepared vector was transformed into BL21DE3, each cellulase was expressed in the cells, and the target cellulase was purified from the cell disruption solution using a nickel column. Next, each purified enzyme was added to the cells washed with 50 mM citrate buffer (pH 5.0) of multi-cohesin yeast cultured overnight in YPD medium, and a binding reaction was performed overnight at 4 ° C. The cells after the binding reaction were washed with 50 mM citrate buffer (pH 5.0), and the PSC degradation activity of CtCel8A-RfDoc (mut) and the Avicel degradation activity of CtCbhA-RfDoc (mut) were obtained in the same manner as in Example 12. It was measured. The results are shown in FIGS. As shown in these figures, 12 cohesins had the highest cellulolytic activity when either purified cellulase was bound. From the results of Examples 12 and 13, it was found that the optimal length when the ScaB cohesin derived from luminococcus flavfaciens was presented to the surface of S. cerevisiae was 12 cohesin.

(Evaluation of proximity effect by cohesin length)
Two types of cellulases were co-expressed in yeasts presenting 1, 4 and 12 cohesins, respectively, and the proximity effect by cohesin length was compared. Specifically, HXT3 homology region, TDH3 promoter and SSRG upstream of RfDoc (mut) -ThCBD1-Mal4 (L2) and RfDoc (mut) -ThCBD1-TrEG2 (L4) (SEQ ID NO: 74 (SEQ ID NO: 75)) PXB-RfDoc (mut) -ThCBD1-Mal4 (L2) and RfDoc (mut) -ThCBD1-TrEG2 (L4) vectors having a signal sequence, DIT1 terminator downstream, Ble marker and HXT3 homology region were prepared (FIG. 28). ). Similarly, Leu3 homologous region, TDH3 promoter and SSRG signal sequence upstream of RfDoc (mut) -ThCBD1-Mal4 (L2) and RfDoc (mut) -ThCBD1-TrEG2 (L4), DIT1 terminator, G418 marker and PLG-RfDoc (mut) -ThCBD1-Mal4 (L2) and RfDoc (mut) -ThCBD1-TrEG2 (L4) vectors having Leu3 homologous regions were prepared (FIG. 29). The obtained vectors were transformed into yeasts presenting 1, 4, and 12 cohesins, respectively, and yeasts expressing various cellulases were produced by homologous recombination.

  The prepared yeast strain was inoculated into 500 μl of YPD liquid medium (10 g / l yeast extract, 20 g / l peptone, 20 g / l glucose), and cultured at 30 ° C. for 20 hours as a preculture solution. 100 μl of the preculture was newly inoculated into 500 μl of YPD liquid medium and cultured at 30 ° C. for 20 hours for main culture. The cells were collected by centrifugation, and the Avicel degradation test was performed using the cells after washing with 50 mM citrate buffer (pH 5.0). Add 100ul of cells suspended in 200ul of 50mM citrate buffer (pH 5.0) to 100ul of 2% Avicel solution, and after reacting at 40 ° C for 4 hours, reduce the amount of reducing sugar in the supernatant by TZ assay. It was measured. All reactions were performed in triplicate. The results are shown in FIG.

  As shown in FIG. 30, the longer the cohesin length, the higher the synergistic effect of the two types of cellulases, and it was found that the proximity effect was further increased by highly integrating cellulases.

(Removal of N-type sugar chain from ScaB-X Docklin)
ScaB-X dockerin is composed of an X domain (module) whose function is unknown and a dockerin domain. As shown in FIG. 31, there is one N-type glycosylation sequence in the X domain, which is expressed in yeast. It was predicted that a sugar chain was added and steric hindrance occurred due to binding with cohesin. Therefore, a ScaBΔX dockerin from which the X domain was removed from the ScaB-X dockerin was prepared, and a binding test to BYScaE strain (ScaE cohesin presenting strain) was performed using the method of Example 3. In addition, a library with an NNK mutation introduced into the N-type glycosylation sequence of the X domain was prepared, and the binding test to BYScaE strain (ScaE cohesin presenting strain) was similarly performed to modify ScaB-X dockerin. It was.

  As a result, the ScaBΔX dockrin from which the X domain was completely removed completely lost the ability to bind to ScaE cohesin. This suggests that the X domain is important for the binding of ScaB-X dockerin and ScaE cohesin. On the other hand, 40 clones were selected from the library with the NNK mutation introduced, and as a result of the binding test with ScaE cohesin, as shown in FIG. Obtained multiple bodies. As a result of sequence analysis of the upper clone, as shown in FIG. 33, a large number of charged amino acids were selected.

  In order to investigate whether the cohesin or dockrin mutant obtained by removing unnecessary sugar chains obtained in Examples 9 and 10 functions significantly in other cellulases, a cellulase other than ThCBD1-Mal4 (L2) was used. PSC degradation activity was evaluated. Specifically, ThCBD1-TrEG2 (L4), ThCBD1-Mal4 (L2), Talaromyces emersonii-derived CBHI (TeCBHI, SEQ ID NO: 76 (SEQ ID NO: 77), CtCBHA, Aspergillus niger-derived CBHA (AnCBHA, SEQ ID NO: 78 (SEQ ID NO: 78 79)), RfDoc (mut) was fused to Phanerochaete chrysosporium-derived Cel7e (PcCel7e, SEQ ID NO: 80 (SEQ ID NO: 81)), and co-expressed in the yeast displaying mutant No. 3 obtained in Example 10, and PSC The degradation activity was measured and is shown in Fig. 34 as relative activity when the PSC degradation activity of cellulosome yeast having natural cohesin and dockerin is 1.

  As shown in FIG. 34, in all cellulases, the PSC degradation activity was improved by optimizing the sugar chain of dockrin. Cellulosomal yeasts with optimized sugar chains for both dockrin and cohesin have further improved PSC degradation activity, and controlling the amount of sugar chains added to both domains is important for constructing artificial cellulosomes in eukaryotes. It was suggested that it is important.

Claims (23)

An artificial skeletal material for holding one or more target proteins,
As a holding site for the one or two or more target proteins, a backbone protein comprising 6 or more and 20 or less cohesins derived from type III cohesins and having binding activity with type III dockins,
Using ScaA cohesin derived from Ruminococcus flavefaciens (Luminococcus flavefaciens) as the cohesin against ScaA dockrin derived from Ruminococcus flavefaciens (Lumicoccus flavefaciens) as a type III dockin possessed by the target protein,
Using ScaE cohesin derived from Ruminococcus flavefaciens (Ruminococcus flavefaciens) as the cohesin against ScaB-X dockin as a type III dockin possessed by the target protein,
Artificial skeleton material.   The artificial skeletal material according to claim 1, wherein the skeletal protein comprises 8 or more and 20 or less of the cohesins.   The artificial skeletal material according to claim 1 or 2, wherein all of one or more N-type sugar chain modification planned sites of the skeletal protein are maintained. The artificial skeletal material according to claim 1 or 2, wherein at least a part of one or more N-type sugar chain modification planned sites provided in the skeletal protein is substituted in any of the following modes .
(A) Asparagine is substituted with an amino acid residue other than asparagine (b) Two residues downstream of serine or threonine of asparagine are substituted with other amino acid residues other than serine and threonine In the embodiment (a), the amino acid residue other than asparagine is phenylalanine, arginine, glutamic acid, glycine, leucine, serine, histidine, threonine, valine, alanine, lysine, proline, glutamine, tryptophan, cysteine, tyrosine, The artificial skeleton material according to claim 4 , which is any one selected from aspartic acid, serine and methionine. An artificial skeletal material for holding one or more target proteins,
As a retention site for the one or more target proteins, a scaffold protein comprising four cohesins derived from a type III cohesin and having binding activity with a type III dockerin,
Using ScaA cohesin derived from Ruminococcus flavefaciens (Luminococcus flavefaciens) as the cohesin against ScaA dockrin derived from Ruminococcus flavefaciens (Lumicoccus flavefaciens) as a type III dockin possessed by the target protein,
Using ScaE cohesin derived from Ruminococcus flavefaciens (Ruminococcus flavefaciens) as the cohesin against ScaB-X dockin as a type III dockin possessed by the target protein,
An artificial skeletal material in which at least a part of one or two or more N-type sugar chain modification scheduled sites provided in the skeletal protein is substituted in any of the following modes.
(A) Asparagine is substituted with an amino acid residue other than asparagine (b) Two residues downstream of serine or threonine of asparagine are substituted with other amino acid residues other than serine and threonine The artificial skeleton according to any one of claims 1 to 6 , wherein the type III dockerin of the target protein is substituted in any one of the following modes at one or more N-type sugar chain modification sites. material.
(A) Asparagine is substituted with an amino acid residue other than asparagine (b) Two residues downstream of serine or threonine of asparagine are substituted with other amino acid residues other than serine and threonine In the embodiment (a), the amino acid residues other than asparagine are each independently selected from the group consisting of glutamic acid, isoleucine, valine, histidine, arginine, aspartic acid, serine and leucine. The artificial skeleton material according to claim 7 . The artificial skeletal material according to any one of claims 1 to 8 , which is an artificial skeletal material that is expressed in a eukaryotic microorganism and presented on the cell surface. The artificial skeleton material according to claim 9 , wherein the eukaryotic microorganism is yeast. The artificial skeletal material according to any one of claims 1 to 10 ,
One or more target proteins each derived from a type III dockerin and having a dockin binding activity to the cohesin;
With
Using ScaA cohesin derived from Ruminococcus flavefaciens (Luminococcus flavefaciens) as the cohesin against ScaA dockrin derived from Ruminococcus flavefaciens (Lumicoccus flavefaciens) as a type III dockin possessed by the target protein,
Using ScaE cohesin derived from Ruminococcus flavefaciens (Ruminococcus flavefaciens) as the cohesin against ScaB-X dockin as a type III dockin possessed by the target protein,
Composite material. The composite material according to claim 11 , which has substitution of any one of the following modes at one or more N-type sugar chain modification scheduled sites of the dockrin.
(A) Asparagine is substituted with an amino acid residue other than asparagine (b) Two residues downstream of serine or threonine of asparagine are substituted with other amino acid residues other than serine and threonine In the embodiment (a), the amino acid residues other than asparagine are each independently selected from the group consisting of glutamic acid, isoleucine, valine, histidine, arginine, aspartic acid, serine and leucine. The composite material according to claim 12 . The composite material according to claim 11 , wherein the one or more target proteins are one or two or more enzymes. The composite material according to any one of claims 11 to 14 , wherein the one or more target proteins are one or more cellulases. Wherein one or more cellulase comprises Phanerochaete chrysosporium (Fanerokete chrysosporium) -derived cellulase and / or Trichoderma reesei (Trichoderma reesei) -derived cellulase comprising the dockerin the N-terminal side, according to claim 15 Composite material. The composite material according to claim 15 or 16 , wherein the one or more cellulases include a cellulase derived from Clostridium thermocellum including the dockrin on the C-terminal side. Eukaryotic microorganisms having an artificial scaffold material according to the cell surface to claim 1-10. The eukaryotic microorganism which has the composite material in any one of Claims 11-17 in a cell surface layer. A recombinant vector for a eukaryotic microorganism, which retains the DNA encoding the protein of the artificial skeletal material according to any one of claims 1 to 10 . The composite material according to any one of claims 11 to 17 , wherein the one or more target proteins comprise a step of bringing a composite material containing one or more cellulases into contact with a cellulose-containing material. A method for producing a saccharified product from contained materials. 18. The composite material according to any one of claims 11 to 17 , wherein the one or more target proteins assimilate cellulose saccharified product in the presence of the composite material containing one or two or more cellulases and the cellulose-containing material. A useful substance production method using a cellulose-containing material, comprising a step of culturing eukaryotic microorganisms. 18. The composite material according to any one of claims 11 to 17, wherein the one or more target proteins assimilate cellulose saccharified product in the presence of a composite material containing one or two or more cellulases. A method for producing a useful substance using a cellulose-containing material, comprising a step of culturing a microorganism.