JP5343873B2 - Eukaryotic microorganism for producing protein using dockerin-cohesin bond and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a protein by using dockerin-cohesin binding in a eukaryotic microorganism. <P>SOLUTION: There is disclosed a eukaryotic microorganism for producing a protein using dockerin-cohesin binding, wherein the eukaryotic microorganism has following characteristics: (a) one or more sugar chain modification-related genes selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2 and MNN11 have been destroyed. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to the acquisition of a protein using a dockerin-cohesin bond, and in particular, to a eukaryotic microorganism for producing the protein and its use.

  In recent years, as an alternative to finite oil resources, expectations for biomass resources derived from the photosynthesis of plants have increased, and various attempts have been made to use biomass for energy and various materials. In order to effectively use biomass as an energy source and other raw materials, it is necessary to saccharify biomass into a carbon source that can be easily used by animals and microorganisms.

  In order to use cellulose and hemicellulose, which are typical biomasses, an excellent cellulase that saccharifies (decomposes) these is required. As such a cellulase source, a cellulosome produced by some bacteria has attracted attention. Cellulosome is a complex of cellulase formed on the surface layer of bacteria and cellulase of skeletal protein (scaffoldin protein) to which the cellulase binds. Scaffoldin protein is a protein having a site called cohesin, and cellulosome is known to be composed of cellulase bound to cohesin in the scaffoldin protein via a domain called dockerin that cellulase has. According to such cellulosome, various cellulases can be provided at high density and in large quantities on the surface of bacterial cells.

  Attempts have been made to construct artificial cellulosomes on the surface of microorganism cells by genetic engineering (Patent Document 1). Patent Document 1 discloses that an artificial cellulosome is constructed on the surface of a yeast cell. By constructing the cellulosome on the cell surface of the yeast, there is an advantage that the cellulase of the cellulosome decomposes the cellulose, and the resulting glucose can be immediately used as a carbon source by the yeast to produce a useful substance by fermentation.

  In constructing an artificial cellulosome, it is preferable to secrete a large amount of cellulase in yeast or the like. For example, regarding the promotion of cellulase secretion, it is disclosed that the secretory production amount is improved by destroying a plurality of N-type sugar chain-modifying enzymes (Patent Document 2). In addition, it has also been disclosed that the amount of antibody production and the ability to form aggregates are improved by adding an O-type sugar chain-modifying enzyme PMT inhibitor and culturing the antibody in methanol-utilizing yeast. (Non-Patent Document 1).

JP2009-142260 JP 2006-280253 A

K. Kuroda et al., Appl. Environ. Microbiol. 74 (2), 446-453 (2008).

  Cellulosomes can be artificially constructed even in eukaryotic microorganisms such as yeast, but further improvement in saccharification ability is required by constructing more highly functional cellulosomes. The binding between the scaffolding protein and cellulase in the cellulosome is thought to depend on the dockerin-cohesin binding properties, but in proteins that use the dockerin-cohesin binding of cellulase and the scaffolding protein, there is a gap between dockerin and cohesin. No attempt has been made to improve the ability of the binding to act (dockerin-cohesin binding) (dockerin-cohesin binding ability), and no such disclosure has been made.

  Then, the indication of this specification aims at acquiring the protein using the dockerin-cohesin coupling | bonding which the binding property to a cohesin or a pair dockerin improved using eukaryotic microorganisms, such as yeast.

  The present inventors paid attention to sugar chain modification among the systems peculiar to eukaryotic microorganisms. In eukaryotic microorganisms such as yeast, there are several sugar chain modification systems. In addition to the possibility that the sugar chain modification to the foreign dockerin protein expressed in the host is involved in the binding to cohesin, the possibility that the sugar chain modification to the cohesin protein is involved in the binding to dockerin And examined. As a result, it was found that the binding property of dockerin to cohesin and the binding property of cohesin to dockerin can be improved by destroying a specific sugar chain modification-related gene. According to the disclosure of the present specification, the following means are provided.

According to the disclosure herein, a eukaryotic microorganism for producing a protein that utilizes dockerin-cohesin binding, comprising the following features:
(A) One or more sugar chain modification-related genes selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 A eukaryotic microorganism is provided comprising: Also provided is the eukaryotic microorganism selected from the group consisting of sugar chain modification-related genes ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11. The protein may be a protein having dockerin, and the protein having dockerin may be a cellulase. Further, the protein may be a protein having cohesin, and the protein having cohesin may be a scaffoldin protein. The eukaryotic microorganism may be a yeast.

In addition, the following features:
(B) Producing a eukaryotic microorganism comprising producing a protein utilizing one or more dockerin-cohesin bonds. The protein using the dockerin-cohesin bond may be a protein having dockerin. The dockerin is derived from one or more microorganisms selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyicum and Ruminococcus flavefacience, The eukaryotic microorganism is also provided.

  The protein using the dockerin-cohesin bond may be a protein having cohesin. The cohesin is derived from one or more microorganisms selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyicum and Ruminococcus flavefacience. Can do.

  According to the disclosure of the present specification, a method for producing a protein using a dockerin-cohesin bond, comprising one or more DNAs encoding the protein using the dockerin-cohesin bond, and CAX4, ALG5, ALG3 One or more glycosylation-related genes selected from the group consisting of ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 are inactivated, Culturing a eukaryotic microorganism to produce the protein.

  According to the disclosure of the present specification, there is provided a method for producing a protein complex, comprising one or more first proteins using dockerin-cohesin, and CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, One or more glycosylation-related genes selected from the group consisting of DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 are inactivated, and the first protein binds to dockerin-cohesin. Contacting the second protein produced using a eukaryotic microorganism carrying DNA encoding one or more second proteins utilizing a dockerin-cohesin bond having a binding domain, Obtaining a complex of a first protein and the second protein.

  In the production method described above, the first protein is selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11. One or more sugar chain modification-related genes may be inactivated and produced using a eukaryotic microorganism that retains the DNA encoding the first protein.

  Either the first protein or the second protein may be displayed on the surface of the eukaryotic microorganism that produces these proteins. In this case, the other protein may be secreted and expressed outside of the eukaryotic microorganism that produces the other protein.

  The one protein may be a protein having cohesin, and the other protein may be a protein having the dockerin. The first protein may be co-expressed with the second protein in a eukaryotic microorganism that produces the second protein.

According to the disclosure herein, the following features:
(A) One or more sugar chain modification-related genes selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 Is inactivated,
(B) producing one or more proteins having cellulase activity and having dockerin; and (c) producing one or more proteins having cohesin.
A method for producing a useful substance is provided, which comprises a step of saccharifying and fermenting a cellulose-containing material using a eukaryotic microorganism.

It is a figure which shows the vector for producing the host yeast for cell surface display. It is a figure which shows the protein gene which has various cloned cohesins. It is a figure which shows the vector for producing the yeast which displays the cohesin containing the protein gene which has various cohesins on the cell surface layer. It is a figure which shows the vector for producing the yeast which carries out secretion production of the dockerin protein (cellulase). It is a figure which shows the evaluation result of the CMC decomposition | disassembly activity in the yeast which the dockerin-cellulase protein secreted and produced in the yeast by which 40 types of sugar chain modification enzymes were each destroyed. It is a figure which shows the evaluation result of the presentation amount in the yeast of the dockerin-cellulase protein secreted and produced in the yeast by which 15 types of sugar chain modification enzymes were each destroyed. It is a figure which shows the evaluation result of the CMC decomposition | disassembly activity of the dockerin-cellulase protein secreted and produced by the ALG6 gene disruption yeast couple | bonded with the cell surface display yeast with the protein which has a some cohesin. It is a figure which shows the result of SDS-PAGE of the dockerin-cellulase protein secreted and produced by the ALG6 gene disruption yeast. It is a figure which shows the vector for carrying out chromosome introduction | transduction by the homologous recombination in the HXT gene which codes the dockerin-cellulase protein containing the dockerin derived from C.cellulolyticum or the dockerin derived from R. flavefaciens. It is a figure which shows the evaluation result of the presentation amount of the dockerin-cellulase protein in the co-expression ALG6 nondestructive or destruction yeast of cohesin protein and dockerin protein. It is a figure which shows the evaluation result of the CMC decomposition | disassembly activity of the dockerin-cellulase protein in the co-expression ALG6 non-destruction or destruction yeast of cohesin protein and dockerin protein.

  The present disclosure relates to the production of proteins using dockerin-cohesin bonds, eukaryotic microorganisms suitable for the production of such proteins, and methods for producing the same, methods for producing proteins using dockerin-cohesin bonds, dockerin-cohesin bonds, and the like. The present invention relates to a method for producing a protein complex utilizing the above and a method for producing a useful substance.

  In eukaryotic microorganisms such as yeast, various proteins such as enzymes involved in various sugar chain modifications are known. Approximately 66 types of sugar chain modifying enzymes in yeast such as Saccharomyces cerevisiae are known. Among them, there are 44 types of sugar chain modifying enzymes that can survive even after gene disruption, including 4 types of sugar chain modifying enzymes that cannot grow upon disruption. The present inventors obtain yeasts in which genes of 40 kinds of sugar chain-modifying enzymes are inactivated, and produce dockerin protein and cohesin protein, which are proteins using dockerin-cohesin bond, to produce cohesin or The binding property to dockerin was examined. As a result, it was found that inactivation of specific 15 kinds of sugar chain-modifying enzyme genes is effective for improving the binding to cohesins such as dockerin proteins. It has not been known so far that these sugar chain-modifying enzymes or genes are genes involved in dockerin-cohesin binding.

  According to the disclosure of the present specification, when a protein utilizing a dockerin-cohesin bond is synthesized by inactivating a specific sugar chain modification-related gene of a eukaryotic microorganism, the binding of the protein The binding property to the cohesin or dockerin as the site can be improved. Such an improvement in binding ability is extremely effective in, for example, expressing proteins that use dockerin-cohesin bonds in eukaryotic microorganisms such as yeast and accumulating complexes of these proteins outside the cells. That is, it is possible to obtain a cohesin protein-dockerin protein complex in which a protein utilizing a dockerin-cohesin bond is accumulated at a larger accumulation amount and / or a higher accumulation degree (accumulation density).

  When the dockerin protein has a predetermined activity such as an enzyme activity, the dockerin protein and the cohesin protein can be combined to increase the activity by bringing the dockerin protein into contact with the cohesin protein. . For example, when the cohesin protein has a plurality of cohesins and the dockerin protein has one dockerin, the plurality of dockerin proteins can be integrated on the cohesin protein. In such a complex, since a large accumulation amount and a higher accumulation degree can be obtained, the activity of the dockerin protein can be effectively enhanced.

  For example, by bringing a dockerin protein having a cellulase active site into contact with a cohesin protein having one or more (preferably a plurality) cohesins, a plurality of dockerin proteins are complexed on the cohesin protein. A cellulase complex (artificial cellulosome) with improved cellulose resolution and saccharification ability can be obtained without greatly depending on the type of cellulase used and its activity. When such a cellulase complex is constructed on the cell surface of a eukaryotic microorganism, the cellulose-containing material can be directly decomposed and saccharified to give the eukaryotic microorganism the ability to directly use cellulose as a carbon source. For example, when the dockerin protein is accumulated in a cohesin protein, the cohesin protein may be co-expressed in a eukaryotic microorganism in which the dockerin protein is expressed, or obtained from another eukaryotic microorganism or the like. It may be supplied to the cell surface of a eukaryotic microorganism whose surface is modified. Hereinafter, the disclosure of this specification will be described in detail.

(Eukaryotic microorganism for producing protein using dockerin-cohesin bond)
In the eukaryotic microorganism disclosed in the present specification, a specific gene is inactivated. Such eukaryotic microorganisms can be used as host cells for producing proteins that utilize dockerin-cohesin bonds. Using this eukaryotic microorganism as a host cell, DNA encoding a protein that uses a dockerin-cohesin bond is introduced and retained so that the dockerin protein can be expressed, thereby producing a protein that uses the dockerin-cohesin bond. be able to.

(Protein using dockerin-cohesin bond)
The protein using the dockerin-cohesin bond disclosed in the present specification includes a protein having at least one of dockerin and cohesin so that the binding ability between dockerin and cohesin can be used. Typically, a dockerin protein having at least one dockerin and a cohesin protein having at least one cohesin are mentioned. The dockerin-cohesin protein also includes a protein having at least one dockerin and at least one cohesin.

(Dockerin protein)
The dockerin protein disclosed in the present specification can include a known dockerin derived from a cellulosome constituent protein or a variant thereof. Examples of dockerin include dockerin domains provided in a part of cellulase as a cellulosome-constituting protein. Since dockerin protein is not inherently intrinsic to eukaryotic microorganisms, it becomes a foreign protein.

  Many dockerins (dockerin domains) that can be possessed by dockerin proteins are known, for example, derived from cellulosome-producing microorganisms listed in Table 1 below. The dockerin domain contained in the cellulosome constituent protein of such a cellulosome-producing microorganism can be appropriately selected or used in combination of two or more. Among them, dockerin derived from a bacterium selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyicum and Ruminococcus flavefacience is preferable.

  The dockerin in the dockerin protein is preferably derived from a cellulosome-producing microorganism of the same kind or the same genus as the cellulosome from which the scaffolding protein to be accumulated is derived. This is because the dockerin domain of the constituent protein of cellulosome has a high binding property with the cohesin domain in the scaffoldin protein in the same or the same genus cellulosome-producing microorganism. More preferably, the dockerin domain is derived from the same type of cellulosome-producing microorganism.

  The dockerin protein can have an active site in addition to dockerin. The type of active site is appropriately determined according to the application. The dockerin protein may be an artificial protein in which dockerin and an active site are appropriately combined. In addition, for example, a cellulase that is a constituent protein of cellulosome, and a cellulase originally having dockerin can be used as it is or after being appropriately modified.

  Dockerin protein can be provided with various enzyme active sites, such as cellulase, when saccharifying and utilizing a cellulose-containing material derived from biomass. As such an active site, an active site in a known cellulase can be appropriately used. 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). You may combine the same or different cellulases classified into a different family.

  Although it does not specifically limit as a cellulase, It is preferable that it is a cellulase with high activity itself. Such cellulases include, for example, Phanerochaete bacteria, Trichoderma bacteria such as Trichoderma reesei, Fusarium bacteria, Tresartes bacteria, Penicillium bacteria, Humicola bacteria In addition to filamentous fungi such as (Humicola), Acremonium, and Aspergillus, Clostridium, Pseudomonas, Cellulomonas, and Luminococcus Bacteria such as genus (Ruminococcus) and Bacillus, primordial fungi such as Sulfolobus, and more, such as Streptomyces and Thermoactinomyces Derived cellulase. Such cellulase or its active site may be artificially modified.

  The dockerin protein may be provided with a hemicellulase active site in consideration of effective utilization of biomass. 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 dockerin protein preferably has extracellular secretion in a eukaryotic microorganism. That is, it is preferably a protein produced as a secreted protein in a eukaryotic microorganism. Enzymes such as cellulase often inherently have a signal for extracellular secretion. A known secretion signal can be used to impart extracellular secretion to the dockerin protein. The secretion signal is appropriately selected according to the type of eukaryotic microorganism used. The secretion signal and the like will be described later.

(Cohesin protein)
The cohesin protein disclosed herein can comprise one or more cohesins that bind dockerin. Cohesin is known as a domain that binds a catalytically active cellulase and the like provided in a type I skeletal protein in a cellulosome formed by a cellulosome-producing microorganism (Nonkan 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.). As the cohesin, a type I cohesin domain on a cellulosomal type I skeletal protein, a type II cohesin domain on the same type II skeletal protein, and a type III cohesin domain on a type III skeletal protein can be used. Many of these types of cohesin domains have been sequenced in various cellulosome-producing microorganisms. The amino acid sequences and DNA sequences of these various types of cohesins are various protein databases and DNA sequence databases accessible via NCBI HP (http://www.ncbi.nlm.nih.gov/), etc. Can be obtained more easily.

  Examples of cohesins included in the cohesin protein include type I and type II of skeletal proteins such as Clostridium thermocellum, Clostridium cellulolyicum, and Ruminococcus flavefacience. And type III cohesin domains and the like can be used.

  The cohesin protein preferably has extracellular secretion or cell surface presentation in eukaryotic microorganisms. That is, it is preferably a protein produced as a secreted protein in a eukaryotic microorganism, or a protein presented on the cell surface of a eukaryotic microorganism. In order to impart extracellular secretion or cell surface presentation property to the cohesin protein, a known secretion signal or surface layer presentation system can be used.

  Cohesin protein is modified by introducing one or more mutations (additions, insertions, deletions and substitutions) in the amino acid sequence of the cohesin as long as it has the binding properties of natural cohesin or dockerin protein derived from a cellulosome-producing microorganism. A cohesin may be provided. The cohesin protein may include a plurality of such cohesins and the like at appropriate intervals. As the whole amino acid sequence of the cohesin protein and the type and presence of the amino acid sequence between cohesins, the amino acid sequence of the type I skeletal protein and a sequence obtained by appropriately mutating the amino acid sequence can be used.

  The cohesin protein preferably has a cellulose binding domain (CBD) of a skeletal 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 (as described above). One or more cellulose-binding domains may be present. Many of the amino acid sequences and DNA sequences of CBD 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.

  A person skilled in the art can produce a cohesin protein having such various domains as appropriate by gene recombination in an appropriate host microorganism. Such a cohesin protein having a cohesin domain of various skeletal proteins can also be obtained by chemical synthesis.

(Glycosylation-related genes to be inactivated)
The eukaryotic microorganism disclosed herein is selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 Alternatively, two or more sugar chain modification-related genes are inactivated. There are various genes related to glycosylation, but in eukaryotic microorganisms, one or more selected from these 15 genes are inactivated and produced in these eukaryotic microorganisms. The binding property of dockerin protein to cohesin and the binding property of cohesin protein to dockerin can be improved.

  These sugar chain modification-related genes are related to N-type sugar chain modification and / or O-type sugar chain modification in yeast. Therefore, if there is an N-type sugar chain modification site or an O-type sugar chain modification site in any of the dockerin and cohesin that are endogenous to or added to the protein to be complexed, and in the vicinity thereof, either of these gene groups By destroying, the dockerin-cohesin binding ability can be improved. That is, the cohesin binding ability of the dockerin protein and the dockerin binding ability of the cohesin protein can be improved. According to the destruction of these sugar chain modification-related genes, for example, dockerin-cohesin binding property can be improved by 1.5 times or more. Especially, it is preferable that it is 1 type, or 2 or more types selected from ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11. By destroying any of these, the dockerin-cohesin binding ability can be improved more than twice.

  The nucleotide sequence and amino acid sequence of each of these sugar chain modification-related genes can be obtained from the Saccharomyces genome database (http://www.yeastgenome.org/), particularly for Saccharomyces genera such as S. cerevisiae. Moreover, about these genes in eukaryotic microorganisms including the genus Saccharomyces, the blast search site of the National Institutes of Health HP (http://www.ncbi.nlm.nih.gov/) (http: // www. ncbi.nlm.nih.gov/blast/Blast.cgi) etc. A strain in which the above-mentioned various genes are disrupted in yeast can be obtained from Open Biosystems (USA).

  As used herein, “a gene is inactivated” refers to a state in which the activity of a protein encoded by the gene is suppressed or completely inhibited. For example, by any manipulation on the gene, transcription and / or translation of the gene is completely or partially inhibited, and the protein encoded by the gene is not produced or quantitatively suppressed, or An embodiment in which a mutant protein in which the original activity of the protein is absent or reduced is included is included. Furthermore, a genetic modification such as administration of a compound that inhibits the activity of the protein encoded by the gene, or inactivation of a protein necessary for transcription or translation, etc. Is also included.

  The eukaryotic microorganism disclosed in the present specification can improve its ability to bind to cohesin as described above when expressing a protein that utilizes dockerin-cohesin binding. Improvement can be achieved by, for example, supplying dockerin protein to an appropriate cohesin protein, and binding amount of dockerin protein as a control obtained under the same conditions except that the specific gene is not inactivated. It can be evaluated by comparing with. The amount of dockerin protein bound can be detected by, for example, adding a fluorescent protein domain or the like to the dockerin protein, so that the dockerin protein involved in the binding to cohesin can be detected.

  An improvement in the ability to bind to cohesin may be determined by measuring the activity specific to the dockerin protein bound to the cohesin protein. For example, when the dockerin protein has a cellulase active site, the cellulase activity may be compared with the control dockerin protein as described above. Specifically, for example, when the dockerin protein has a cellulase active site such as endoglucanase, an appropriate cellulase substrate (carboxymethylcellulose, phosphate) is used for the collected culture supernatant or the eukaryotic microorganism displaying the dockerin protein on the surface. The enzyme activity can be evaluated by measuring the amount of the reaction product, the base mass and the like by reacting with cellulose, crystalline cellulose and the like. The reaction temperature, pH, and time can be appropriately set depending on the type of enzyme. In addition, methods for quantifying the amount of reducing sugar resulting from the enzyme reaction include the Somogyi method, Tauber-Kleiner method, Hanes method (titration method), Park-Johnson method, 3,5-dinitrosalicylic acid (DNS) method, TZ method (Journal of Biochemical Methods, 11 (1985) 109-115) or the like may be employed as appropriate. Furthermore, cellulase activity such as endoglucanase is supplied to the evaluation region consisting of a solid phase containing cellulose such as carboxymethyl cellulose, and the test protein that has the potential as the present invention is supplied. It can also be evaluated by the size of a region where the cellulose is decomposed and disappeared in the solid phase (halo: a region where the solid phase is lightened or colorless due to decomposition of biomass).

  The eukaryotic microorganism disclosed in the present specification can improve its ability to bind dockerin when cohesin protein is similarly expressed. In that case, the binding ability of the cohesin protein to dockerin can be confirmed according to the method for confirming the binding ability of the dockerin protein to cohesin.

  In order to inactivate a specific sugar chain modification-related gene, for example, such an endogenous gene may be knocked out. Knockout of a specific gene can be easily performed by those skilled in the art. For example, a DNA fragment having a homologous sequence capable of homologous recombination with the protein coding region of a specific gene on the chromosome or the vicinity thereof is prepared and introduced into a eukaryotic cell to cause homologous recombination in the cell. Can be realized. The introduction of a DNA fragment for knockout into a eukaryotic microorganism will be described later.

  Eukaryotic microorganisms disclosed herein are suitable host microorganisms for producing proteins that utilize dockerin-cohesin bonds, typically as foreign proteins. Such eukaryotic microorganisms are not particularly limited, and various known yeasts can be used, for example. 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.

(Eukaryotic microorganisms that produce proteins that use dockerin-cohesin bonds)
The eukaryotic microorganism disclosed herein may produce a protein that utilizes one or more dockerin-cohesin bonds. That is, one or two or more DNAs corresponding to such proteins may be retained so that each protein can be expressed.

  The eukaryotic microorganism disclosed herein may be one that produces one or more dockerin proteins. That is, DNA encoding one or more dockerin proteins may be introduced and retained so that the dockerin protein can be expressed. The dockerin protein produced by such eukaryotic microorganisms can be expected to improve the active site of the protein as well as binding to cohesin. Two or more dockerin proteins are distinguished by the kind and number of dockerin domains and the form of the active site.

  Moreover, the eukaryotic microorganism disclosed in the present specification may be one that produces one or more cohesin proteins. That is, a DNA encoding one or more cohesin proteins may be introduced and retained so that the cohesin protein can be expressed. In addition, the cohesin of the cohesin protein is preferable for the dockerin protein. Therefore, such a eukaryotic microorganism is a preferable host for accumulating dockerin protein to cohesin protein outside the cell. In addition, by simultaneously expressing the dockerin protein and the cohesin protein in the eukaryotic microorganism, a complex of the cohesin protein and the dockerin protein can be constructed outside the cell. In particular, when cohesin protein is displayed on the cell surface, a complex of these proteins can be constructed on the cell surface. Two or more cohesin proteins are distinguished by the type and number of cohesin domains and the form of the active site.

  Further, the eukaryotic microorganism disclosed herein may express both one or more dockerin proteins and one or more cohesin proteins.

  The encoded DNA encoding the protein is not particularly limited as long as the protein can be expressed in the host microorganism so that the protein can be expressed. 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 general, with the introduction of such foreign DNA, a selectable marker gene that can be used in the host is also retained.

  It is preferable to impart extracellular secretion and cell surface presentation to dockerin protein and cohesin protein produced in eukaryotic microorganisms. Among them, it is preferable to impart extracellular secretion to the dockerin protein, and it is preferable to impart cell surface presentation to the cohesin protein that is further secreted extracellularly and presented on the cell surface. In order to confer extracellular secretion, a secretion signal can be imparted. 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. Further, by using an aggregating protein or a part thereof, the protein can be secreted into a state presented on the surface layer of a eukaryotic microorganism. For example, there is a peptide consisting of 320 amino acid residues in the 5 'region of the SAG1 gene encoding α-agglutinin which is an aggregating protein. 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.

  The eukaryotic microorganisms that produce eukaryotic microorganisms or proteins that use dockerin-cohesin bonds as the hosts disclosed in the present specification described above are molecular cloning 3rd edition, Current Protocols in Molecular Biology, etc. It can be produced according to the method described in 1. For example, a strain in which a specific gene is disrupted in yeast can be obtained as appropriate, and a vector for inactivating one or more genes selected from the disclosed specific glycosylation-related genes can be It may be inactivated by introduction into a nuclear microorganism. Such vectors and their construction methods 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. Similarly, vectors for expressing dockerin protein and scaholdin protein in eukaryotic microorganisms and methods for constructing them are also well known to those skilled in the art. 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.

  The eukaryotic microorganism disclosed in the present specification can be obtained by transforming a eukaryotic microorganism with such a vector. For the transformation, 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. In addition, the eukaryotic microorganism that produces a protein that utilizes the dockerin-cohesin bond disclosed in the present specification is an eukaryotic microorganism in which one or more specific glycosylation-related genes are destroyed. It is preferably produced by introducing an expression vector for a protein that utilizes a dockerin-cohesin bond, such as a dockerin protein and a cohesin protein.

(Producing method of protein using dockerin-cohesin bond)
A method for producing a protein using a dockerin-cohesin bond disclosed in the present specification comprises a DNA encoding the protein using the dockerin-cohesin bond, and includes CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, A protein obtained by culturing a eukaryotic microorganism in which one or more glycosylation-related genes selected from the group consisting of DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 are inactivated. A step of producing
By culturing a eukaryotic microorganism having a DNA encoding a dockerin-cohesin protein and capable of expressing the protein, wherein the specific sugar chain modification-related gene is inactivated, the dockerin-cohesin binding ability Can improve the protein. The culture conditions for eukaryotic microorganisms can be appropriately set by those skilled in the art depending on the type of microorganism. Further, liquid culture using a liquid medium is preferable, but the culture method is not particularly limited. When a protein using dockerin-cohesin bond has extracellular secretion, the protein can be obtained in the culture supernatant obtained in the culture step.

(Producing method of protein complex)
The protein complex production method disclosed in the present specification includes a first protein using dockerin-cohesin, CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2 , ANP1, MNN2, and MNN11, one or two or more glycosylation-related genes are inactivated and have a dockerin-cohesin bond having a domain that binds to the first protein by a dockerin-cohesin bond A complex of the first protein and the second protein is obtained by contacting with a second protein produced using a eukaryotic microorganism that holds DNA encoding the second protein to be used. The step of performing. Here, the 1st protein and the 2nd protein should just have dockerin and / or cohesin, respectively so that both can be combined by dockerin-cohesin bond.

  The first protein may also be produced using a eukaryotic microorganism in which one or more of the specific genes are inactivated and the DNA encoding the first protein is retained. By doing so, the first protein also has an improved dockerin-cohesin binding ability and can easily form a protein complex.

  Either one of the first protein and the second protein may be presented on the cell surface of a eukaryotic microorganism that expresses the protein. In this case, the protein complex can be presented on the surface layer of the eukaryotic microorganism presenting the one protein by secreting and expressing the other protein outside the cell. In this case, this method takes the form of a method for producing a eukaryotic microorganism having a protein complex on the cell surface.

  When the first protein and the second protein are expressed in the present eukaryotic microorganism, they may be expressed in separate eukaryotic microorganisms. In this case, when both proteins are secreted and expressed outside the cell, a protein complex can be constructed outside the cell by co-culturing these microorganisms. By allowing one protein to be presented on the cell surface and the other to be secreted extracellularly, a protein complex can be constructed on the cell surface of eukaryotic microorganisms by culturing these microorganisms. In addition, it is preferable that these microorganisms are suitable for simultaneous culture. That is, it is preferably a closely related species or an eukaryotic microorganism of the same species. These proteins may be co-expressed in one eukaryotic microorganism. In this case, the protein complex can be constructed extracellularly or on the cell surface layer by selecting the expression form of each protein outside the cell as described above.

  Further, one of the first protein and the second protein is a cohesin protein having a plurality of cohesins, and the other is a dockerin, preferably one dockerin, less than the number of cohesins of the first protein. By using a dockerin protein having a protein, a protein complex in which a plurality of dockerin proteins are complexed on a cohesin protein can be obtained. Furthermore, in this embodiment, such a complex can be easily expressed on the surface of the eukaryotic microorganism by expressing the dockerin protein in the extracellular layer of the eukaryotic microorganism so that the cohesin protein is presented and expressed on the cell surface of the eukaryotic microorganism. Can present.

  The method for contacting the first protein and the second protein is not particularly limited. What is necessary is just to mix both etc. in the liquid of pH, salt concentration, and temperature which protein does not denature. Both proteins may be present in the liquid as free proteins. The other may be presented on the cell surface. It may be in a culture solution of a eukaryotic microorganism that expresses the first protein and / or the second protein.

  When the dockerin protein has cellulase activity, a protein complex in which cellulase is accumulated on the cell surface of a eukaryotic microorganism can be constructed. Such eukaryotic microorganisms can use glucose obtained by degrading saccharification of a cellulose-containing material on the cell surface as a carbon source.

(Useful substance production method)
The method for producing a useful substance disclosed in the present specification has the following characteristics;
(A) One or more sugar chain modification-related genes selected from the group consisting of CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2, and MNN11 Is inactivated,
(B) producing a dockerin protein having cellulase activity; and (c) producing a cohesin protein.
And a step of saccharifying and fermenting the cellulose-containing material using a eukaryotic microorganism. According to this method, the cellulose-containing material can be directly decomposed and saccharified using this eukaryotic microorganism and used as glucose or the like. By carrying out the fermentation step, useful substances are produced according to the useful substance production capacity of the eukaryotic microorganism used.

  A useful substance is a product obtained by fermenting glucose or the like by a eukaryotic microorganism, and varies depending on the type of eukaryotic microorganism and also on 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. 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.

  As the dockerin protein having cellulase activity used in the present method, it is preferable to use two or more types each having two or more types of cellulase (for example, endoglucanase and cellobiohydrolase) activities. The dockerin protein may be co-expressed in a single eukaryotic microorganism or may be independently expressed in two or more eukaryotic microorganisms.

  The cellulose-containing material is a material containing cellulose which is β-glucan in which D-glucose is glycosidically bonded with β-1,4 bonds. As a cellulose containing material, what is necessary is just to contain a cellulose, and what kind of origin and form may be sufficient as it. 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.

  Prior to contact with the cellulase, the cellulose-containing material may be subjected to an appropriate pretreatment or the like in order to facilitate degradation by the cellulase. For example, cellulose can be made amorphous or low molecular by partially hydrolyzing cellulose under acidic conditions with inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. In addition, the cellulose can be made amorphous or low molecular by treatment with supercritical water, alkali, pressurized hot water, or the like.

  The cellulose-containing material includes a polymer in which glucose is polymerized by β-1,4-glycoside bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. In addition, examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. The cellulose may be crystalline cellulose or non-crystalline cellulose.

  Through the various embodiments described above, when terms such as eukaryotic microorganisms, proteins using dockerin-cohesin bonds, dockerin proteins, cohesin proteins, sugar chain modification-related genes, cellulases, cellulose-containing materials are used. These are used in common with the contents defined in this specification.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination described below was performed according to Molecular Cloning: A Laboratory Manual (T. Maniatis, et al., Cold Spring Harbor Laboratory).

(Preparation of host yeast for cell surface display)
A pAI-HOR7p-AGA1 vector (FIG. 1) having an AAP1 homologous region and a HOR7 promoter upstream of the aga1 gene cloned according to a conventional method by PCR and a Tdh3 terminator, His3 marker and AAP1 homologous region downstream was prepared. Using this vector, yeast S. cerevisiae BY4741 was transformed to obtain yeast BY-AGA1, which displays a large amount of aga1 on the cell surface.

(Production of yeast displaying cohesin protein on the cell surface)
Cohesin and CBD-cohesin, which are elements of the cohesin protein, were cloned from three types of cellulosome-producing microorganisms (see FIG. 2). Cohesin (Ctcoh) (SEQ ID NO: 1) and CBD-4 cohesin (CtCBD4coh) (SEQ ID NO: 2) were amplified from the sequence of C. thermocellum-derived skeletal protein cipA by PCR according to conventional methods and cloned (FIG. 2 (a) )). Similarly, cohesin (Cccoh) (SEQ ID NO: 3) was cloned from the sequence of C. cellulolyticum-derived skeletal protein cipC by PCR according to a conventional method and then cloned (FIG. 2 (b)). Further, from the sequence of the R. flavefaciens-derived skeletal protein ScaB, cohesin (Rfcoh) (SEQ ID NO: 4) was obtained by gene synthesis (FIG. 2 (c)).

  PDL-HOR7p-CtCoh4GAh2 vector, pDL-HOR7p-CtCBD4CohAGA2 vector, pDL-HOR7p-CtCBD4CohAGA2 vector, ADH3 homology region and HOR7 promoter upstream of each acquired gene, V5-tag, aga2, Tdh3 terminator, Leu2 marker and ADH3 homology region downstream -HOR7p-CcCohAGA2 vector and pDL-HOR7p-RfCohAGA2 vector were prepared (FIG. 3). By introducing these vectors into BY-AGA1 obtained in Example 1, yeast Ctcoh and CtCBD4coh presenting C. thermocellum-derived cohesin on the cell surface, yeast Cccoh presenting C. cellulolyticum-derived cohesin on the cell surface, and R Each yeast Rfcoh presenting flavefaciens-derived cohesin on the cell surface was obtained.

(Production of yeast producing secretory production of dockerin-cellulase protein)
From the genome of C. thermocellum, the Cel8A cellulase gene (SEQ ID NO: 5) and the Cel48S dockerin gene (SEQ ID NO: 6) were amplified by PCR according to a conventional method and then cloned. The obtained gene was joined and inserted into a 2 mm vector (FIG. 4) in a form in which a His-tag was added upstream to prepare a 2 mm-Cel8A-Ctdoc vector. The obtained vector was introduced into each sugar chain-modifying enzyme-disrupted strain (Open Biosystems (USA)) shown in Table 2 of yeast S. cerevisiae BY4741, and each sugar chain-modifying enzyme-disrupted Cel8A-Ctdoc secretion-producing yeast was obtained. .

(Evaluation of cellulase activity of dockerin-cellulase protein bound to cohesin protein cell surface display yeast)
The yeast Ctcoh obtained in Example 2 was cultured in YP + 2% glucose medium at 30 ° C. for 24 hours, and 200 ml of OD600 = 10 cells were collected. Each strain obtained in Example 3 was cultured in a YNB + 2% casamino acid + 2% glucose medium at 30 ° C. for 24 hours, 1.5 ml of OD600 = 8 cells were collected, and the culture supernatant was collected. A chain-modified enzyme-disrupted Cel8A-Ctdoc secretion was obtained. The collected Ctcoh is suspended in 1 ml of a large excess of the sugar chain-modified enzyme-disrupted Cel8A-Ctdoc secretion, and left at 4 ° C for 12 hours, so that the yeast with reconstituted Cel8A-Ctdoc and Ctcoh on the cell surface is obtained. I got it. The yeast was collected in an amount equivalent to 1 ml of OD600 = 1, washed with 50 mM acetate buffer pH 6.0, mixed with 1% CMC, 50 mM acetate buffer pH 6.0 solution, and subjected to CMC degradation reaction at 60 ° C. . After the reaction, free reducing sugar was measured by TZ assay. The results are shown in FIG.

  As shown in FIG. 5, 15 types out of 40 types in which the CMC degrading activity was improved were found among the yeasts bound with the sugar chain modifying enzyme-disrupted Cel8A-Ctdoc. That is, CAX4, ALG5, ALG3, ALG9, ALG12, ALG6, LG8, DIE2, OST3, OST5, PMT1, PMT2, ANP1, MNN2 and MNN11 were found.

(Evaluation of cell surface display amount of dockerin-cellulase protein bound to cohesin protein cell surface display yeast)
Yeast Ctcoh bound to the sugar chain-modifying enzyme disrupted Cel8A-Ctdoc obtained in Example 4 with an improved CMC degradation activity was collected at an OD600 = 0.5, equivalent to 62.5 ml, washed with a PBS solution, PBS + It was mixed with 1 mg / ml BSA + anti-His-FITC solution, reacted at 4 ° C for 30 minutes, washed twice with PBS solution, and then the amount of CelA presented on the surface of yeast cells was evaluated by Flow Cytometry. The results are shown in FIG.

  As shown in FIG. 6, 12 types of 15 yeasts in which the amount of Cel8A presented was improved by destroying the sugar chain modifying enzyme were found. That is, ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11 were found. The increase in the amount of dockerin bound to the skeletal protein suggested that the binding property of dockerin to cohesin was improved and the binding of the giant sugar chain to the sugar chain modification site present in dockerin was released.

(Evaluation of cellulase activity of dockerin-cellulase protein in which cohesin protein having a plurality of cohesins is bound to yeast displayed on the cell surface)
The yeast CtCBD4coh obtained in Example 2 was cultured in a YP + 2% glucose medium at 30 ° C. for 24 hours, and 200 ml of OD600 = 10 cells were collected. Cel10A-Ctdoc and CtCBD4coh were reconstituted on the cell surface in the same manner as in Example 4 for 10 of the 12 sugar chain modifying enzyme-disrupted Cel8A-Ctdoc secretion-producing yeasts obtained in Example 5. The CMC degradation activity was evaluated. The results are shown in FIG.

  As shown in FIG. 7, it was proved that cancellation of the sugar chain modification of dockerin is effective even for yeast having a huge scaffold protein (scaffoldin) containing four cohesins and CBD.

(Confirmation of sugar chain modification state in dockerin-cellulase protein secreted and produced by ALG6-disrupted yeast)
Among the yeasts obtained in Example 3, ALG6-disrupted Cel8A-Ctdoc secretion-producing yeast was used with a jar fermenter, YNB + 2% casamino acid + 2% glucose medium, aeration 0.5 vvm, stirring speed 400 rpm, pH 5.0, The culture was performed at 30 ° C. for 24 hours. The culture supernatant was collected by centrifugation, Cel8A-Ctdoc was purified with a His-affinity column, and the molecular weight was evaluated by SDS-PAGE. The results are shown in FIG.

  As shown in Fig. 8, it is one of the sugar chain-modifying enzymes compared to the smear band in the high molecular region found in Cel8A-Ctdoc (W: wild type) produced in yeast that has not destroyed the sugar chain-modifying enzyme. The molecular weight of Cel8A-Ctdoc produced from yeast that disrupted ALG6 was greatly reduced, suggesting that the ALG6 disruption was able to release the giant sugar chain modification added to Cel8A-Ctdoc.

(Production of cohesin-dockerin co-expression yeast)
From the C. cellulolyticum genome, the Cel5A dockerin gene (SEQ ID NO: 7) was amplified by PCR according to a conventional method and then cloned. In addition, a ScaA dockerin gene (SEQ ID NO: 8) was obtained from the R. flavefaciens-derived ScaA sequence by gene synthesis. The obtained genes were joined to the C. thermocellum-derived Cel8A cellulase gene obtained in Example 3, and the HXT3 homology region, HOR7 promoter and His-tag were upstream of each, and the Tdh3 terminator, URA3 marker and HXT3 homology region were downstream. The pXU-HOR7p-Cel8A-Ccdoc vector and the pXU-HOR7p-Cel8A-Rfdoc vector (FIG. 9) were prepared. Each of these vectors was introduced into the two types of yeast Cccoh and Rfcoh obtained in Example 2, respectively, and the yeast Cccoh-Cel8A and R. flavefaciens-derived cohesin that co-produces C. cellulolyticum-derived cohesin and Cel8A and displays them on the cell surface. The yeast Rfcoh-Cel8A, which simultaneously produces Cel8A and presents it on the cell surface, was obtained.

(Preparation of cohesin-dockerin co-expressing ALG6-disrupted yeast)
The disruption region gene was cloned from the genome of the ALG6 disruption strain of yeast S. cerevisiae BY4741 after amplification by PCR according to a conventional method. The obtained gene was introduced into the yeasts Cccoh-Cel8A and Rfcoh-Cel8A obtained in Example 8, and ALG6-disrupted yeast Cccoh-Cel8A and ALG6-disrupted yeast Rfcoh-Cel8A were obtained.

(Evaluation of cell surface display amount of dockerin protein in cohesin-dockerin co-expressing yeast)
Yeast Cccoh-Cel8A and yeast Rfcoh-Cel8A obtained in Example 8 and ALG6 disrupted yeast Cccoh-Cel8A and ALG6 disrupted yeast Rfcoh-Cel8A obtained in Example 9 were each 30 ° C. in YP + 2% glucose medium, Cultivate for 24 hours, collect OD600 = 0.5, 62.5 ml equivalent, wash once with PBS solution, mix with PBS + 1 mg / ml BSA + anti-His-FITC solution, react at 4 ° C for 30 minutes After washing twice with PBS solution, the amount of CelA presented on the surface of yeast cells was evaluated by Flow Cytometry. The results are shown in FIG.

  As shown in FIG. 10, in the combination of C. cellulolyticum-derived cohesin and dockerin derived from the same, and in the combination of R. flavefaciens-derived cohesin and dockerin derived from the same, the ALG6 gene was disrupted to reduce the amount of CelA presented An improvement was observed. The combination of C. cellulolyticum-derived cohesin and the same derived dockerin has an N-type sugar modification site and an O-type sugar chain modification site only on the cohesin side, and no modification site exists on the dockerin side. Therefore, the improvement in the amount of CelA presented in yeast by this combination supports that the ability to bind dockerin has been improved by suppressing or avoiding sugar chain modification to cohesin by disruption of such a gene.

(Evaluation of cellulase activity of dockerin protein in cohesin-dockerin co-expressing ALG6-disrupted yeast)
Yeast Cccoh-Cel8A and yeast Rfcoh-Cel8A obtained in Example 8 and ALG6 disrupted yeast Cccoh-Cel8A and ALG6 disrupted yeast Rfcoh-Cel8A obtained in Example 9 were cultured in YP + 2% glucose medium at 30 ° C. for 24 hours. OD600 = 1, 1 ml equivalent amount was collected, washed with 50 mM acetate buffer pH 6.0 solution, mixed with 1% CMC, 20 mM acetate buffer pH 6.0 solution, and CMC decomposition reaction was performed at 60 ° C. . The results are shown in FIG.

  As shown in FIG. 11, by destroying the sugar chain-modifying enzyme, an improvement in CMC degradation activity was observed, indicating that the saccharification ability of yeast was improved. Considering the degree of improvement in CMC degradation activity, it was found that the cellulase activity of dockerin protein was improved by the destruction of the sugar chain modification-related gene.

Claims (10)

A dockerin protein having cellulase activity or hemicellulase activity having one or more dockerins having a sugar chain modification site and / or one or more cohesins having a sugar chain modification site, from which the dockerin is derived a yeast that produces a cohesin protein with cohesin derived from cellulosome producing microorganism of the same kind and,
The following features:
(A) one glycosylation-related gene selected from the group consisting of ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11 is disrupted; and (b ) the dockerin protein and / or the cohesin protein expressed extracellularly,
Yeast comprising.   The yeast according to claim 1, wherein the cohesin protein and the dockerin protein are coexpressed.   The dockerin protein is derived from one or more microorganisms selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyicum and Ruminococcus flavefacience. The yeast of Claim 1 or 2 which has dockerin.   The cohesin protein is derived from one or more microorganisms selected from the group consisting of Clostridium thermocellum, Clostridium cellulolyicum, and Ruminococcus flavefacience. The yeast according to any one of claims 1 to 3.   The yeast according to any one of claims 1 to 4, wherein the yeast is Saccharomyces cerevisiae. A dockerin protein having cellulase activity or hemicellulase activity having one or more dockerins having a sugar chain modification site and / or one or more cohesins having a sugar chain modification site, from which the dockerin is derived A method for producing a cohesin protein having a cohesin derived from the same kind of cellulosome-producing microorganism,
One glycosylation-related gene selected from the group consisting of ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11 is disrupted, and the dockerin protein and / or the Culturing yeast that expresses cohesin protein extracellularly to produce the dockerin protein and / or the cohesin protein. A method for producing a protein complex comprising:
The complex is a dockerin protein having cellulase activity or hemicellulase activity having one or more dockerins having a sugar chain modification site, and one or more cohesins having a sugar chain modification site, wherein the dockerin A complex with a cohesin protein having a cohesin derived from the same type of cellulosome-producing microorganism from which
Contacting the dockerin protein and the cohesin protein to obtain a complex,
The dockerin protein and / or the cohesin protein is associated with one glycosylation modification selected from the group consisting of ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11 A method produced using yeast in which a gene is disrupted.   The method according to claim 7, wherein the cohesin protein and the dockerin protein are obtained by co-expression in the yeast.   The method according to claim 7 or 8, wherein the cohesin protein is displayed on the surface of the yeast and the dockerin protein is secreted and expressed outside the cell. The following features:
(A) one sugar chain modification-related gene selected from the group consisting of ALG5, ALG3, ALG9, ALG12, ALG6, ALG8, DIE2, OST3, OST5, PMT1, PMT2, and MNN11 is inactivated,
(B) having one or more dockerins having sugar chain modification sites, producing one or more dockerin proteins having cellulase activity or hemicellulase activity, and (c) 1 having sugar chain modification sites Or two or more cohesins that produce one or more cohesin proteins having cohesins derived from the same kind of cellulosome-producing microorganism from which the dockerin is derived.
Saccharifying and fermenting a cellulose-containing material using yeast,
A method for producing useful substances.