JP5119466B2 - Yeast production method, yeast, and glycoprotein or β-glucan production method - Google Patents

Description

  The present invention relates to a method for producing yeast in which high temperature sensitivity is avoided or growth is restored by controlling a proofreading function in a DNA polymerase of a gene function deficient strain such as a gene disrupted strain of yeast and inducing a desirable mutation, And a yeast obtained by using such a production method. More specifically, the present invention uses a budding yeast having a glycoprotein-producing ability having a mammalian sugar chain, or a yeast gene-deficient strain such as fission yeast, and alters the amino acid sequence of the DNA polymerase to induce mutation. It is related with the manufacturing method for obtaining the yeast which avoided the high temperature sensitivity or recovered | restored the proliferative property, etc., or the yeast which has the expression ability of a foreign gene.

  In addition, the present invention produces a polysaccharide (particularly β-glucan) with high efficiency by controlling a proofreading function in a DNA polymerase of a gene function deficient strain such as a yeast gene disruption strain and inducing a desirable mutation. The present invention relates to a yeast production method, and a yeast obtained using such a production method. More specifically, the present invention uses a yeast gene function-deficient strain having a glycoprotein-producing ability having a mammalian sugar chain, alters the amino acid sequence of the DNA polymerase, and induces a mutation. The present invention relates to a production method for obtaining yeast containing a β-glucan used as an active ingredient of a pharmaceutical in a cell wall with high efficiency.

  Many glycoproteins having sugar chains have important functions in vivo. In addition, it has been clarified by erythropoietin (EPO), tissue plasminogen activator (TPA), etc. that a sugar chain is removed from glycoprotein (Kiyoyo “Protein Nucleic Acid Enzyme” Vol. 36, p775 (1991); Makoto Takeuchi “Biochemistry” vol. 62, p1272 (1990)). From this, it can be said that the sugar chain in glycoprotein plays an important role in biological activity. Therefore, effective production of glycoproteins is desirable in developing pharmaceuticals and the like.

  When producing glycoproteins, it is conceivable to use yeast in terms of being able to apply genetics and molecular biological techniques, and being a single-cell eukaryote with a high ability to produce foreign heterologous proteins. On the other hand, since the sugar chain produced by yeast has a structure in which many mannoses are added, it exhibits strong antigenicity in higher animals. For this reason, there exists a problem that the glycoprotein produced by yeast is not necessarily suitable especially for the pharmaceuticals administered transvascularly. In order to solve such problems, yeast strains that produce sugar chains (human-type sugar chains) that have lost antigenicity by gene disruption have been constructed (for example, JP-A-6-277086, JP-A-7-299509). No. and JP-A-2001-161376).

  In particular, an international publication WO01 / 014522 pamphlet (see Patent Document 1 below) discloses a triple disruption strain having och1 disruption (Δoch1), mnn1 disruption (Δmnn1), and mnn4 disruption (Δmnn4). That is, among genes involved in the biosynthesis of an outer sugar chain specific to yeast, a gene (OCH1) encoding α-1,6 mannosyltransferase that performs the first extended addition reaction, mannose at the non-reducing end of the sugar chain The function of the gene (MNN1) encoding α-1,3 mannosyltransferase that adds mannose and the gene (MNN4) that controls addition of mannose-1-phosphate was disrupted, or some mutation was introduced into these genes Yeast variants have been disclosed. Since these yeast strains are excellent in the production of glycoproteins containing mammalian sugar chains, it is considered useful for the development of functional foods and pharmaceuticals.

  However, gene-disrupted strains are more temperature-sensitive than wild-type strains, and are sensitive to high temperatures, such as not growing at 37 ° C., and have poor growth potential. As a result, the gene-disrupted strain has a problem in that it exhibits poor growth compared to the wild strain and further has a low protein production ability.

  By the way, β-glucan, a kind of polysaccharide, activates macrophages, NK cells, T cells, and killer T cells that attack infected cells and cancer cells in the body, and has the effect of enhancing immunity and resistance. It is known to have. This immunity-enhancing action increases the ability to eliminate bacteria and foreign substances that have entered the body, and even if it is infected, it has resistance to suppress the disease. In addition, such an increase in immunity can be expected to suppress allergic reactions and suppress tumors such as cancer. Actually, various clinical trials have revealed antitumor properties. Furthermore, effects such as a decrease in blood glucose level, diuretic action, blood pressure adjustment, and a decrease in blood cholesterol and triglyceride levels are also obtained.

  Yeast (especially baker's yeast) has been used in fermented foods for a long time and is extremely safe as food. Since baker's yeast usually contains about 45% β-glucan in the cell wall, it has been commercialized as a health supplement targeted at an immune enhancing effect. Baker's yeast β-glucan is utilized mainly by extraction from the cell wall. Β-glucan derived from baker's yeast is already sold as a pharmaceutical product in the United States as zymosan.

  In order to obtain more β-glucan from the cell wall of the yeast, it is necessary to culture the yeast in a large amount. Moreover, after culture | cultivation, the highly efficient extraction operation | work of (beta) -glucan is needed, but since this operation requires a special technique, it is not easy. Therefore, it is desired to develop a method capable of producing such yeast-derived β-glucan by omitting such a step as much as possible and further easily and inexpensively.

  On the other hand, a mutagenesis method for inducing mutation by coexisting two or more kinds of DNA polymerases having different fidelity in one E. coli cell is known (Japanese Patent Laid-Open No. 2002-262875).

  In addition, International Publication WO 00/028015 pamphlet (Patent Document 2 below) describes a gene characterized by introducing more point mutations into one strand of a double-stranded genomic DNA of a cell or organism than the other. "Method for introducing mutation into" (Claim 1 of the same publication). In the publication, “by accumulating more random point mutations in one of the DNA strands than in the other, it is possible to increase the mutation rate and reduce the risk of extinction of the mutated cells and living organisms efficiently. "Effectively obtain a mutant" is described (from the bottom of page 9 of the same publication, the third and subsequent lines). However, although there is an example using Escherichia coli in the publication, there is no example using yeast. Therefore, it is not always clear what mutation is induced when the technique disclosed in the publication is applied to yeast.

International Publication WO01 / 014522 Pamphlet International Publication WO00 / 028015 Pamphlet

  As described above, in general, so-called gene function-deficient strains such as yeast gene-disrupted strains or gene mutant strains often have negative traits in viability such as high-temperature sensitivity and reduced growth. It is an object of the present invention to provide a method for producing a yeast that avoids high-temperature sensitivity or restores the growth ability, and a yeast obtained by such a production method.

  The present invention avoids high-temperature sensitivity or restores proliferative properties in gene-deficient strains such as gene-disrupted strains or gene-mutant strains that have the ability to produce glycoproteins having mammalian sugar chains. An object of the present invention is to provide a method for producing a yeast excellent in protein productivity.

  An object of the present invention is to provide a method for producing a glycoprotein having a mammalian sugar chain using the method for producing a yeast that avoids high-temperature sensitivity as described above or restores its growth ability.

  In addition, as described above, since it is desired to develop a method capable of producing yeast-derived β-glucan more easily and inexpensively, the present invention provides a method for producing yeast that produces β-glucan with high efficiency, and its It aims at providing the yeast obtained by such a manufacturing method.

  An object of this invention is to provide the manufacturing method of (beta) -glucan using the manufacturing method of the yeast which produces the above-mentioned (beta) -glucan efficiently.

  An object of this invention is to provide the manufacturing method of the yeast which has the expression ability of a foreign gene, and the manufacturing method of a foreign protein using such a manufacturing method.

  The present invention basically induces a beneficial mutation by controlling the proofreading function of DNA polymerase in a gene function-deficient strain such as a gene disruption strain or a gene mutant strain of yeast. As a result of repeating, while having a mammalian sugar chain, the growth delay was recovered, and a yeast having excellent high-temperature resistance and protein-producing ability and a novel mutant of yeast having excellent β-glucan-producing ability could be produced. Based on experimental findings. That is, the present invention obtains a yeast suitable for protein production and β-glucan production ability by expressing a pol3 gene or cdc6-gene introduced with a mutation involved in the control of the proofreading function in yeast and repeating the culture. It is based on the knowledge that it is possible.

  That is, the first aspect of the present invention is a yeast that avoids high-temperature sensitivity or restores growth properties, including a step of controlling the proofreading function of DNA polymerase in a gene function-deficient strain such as a gene-disrupted strain or gene mutant of yeast. And a method for producing yeast that produces β-glucan with high efficiency. A preferred embodiment of the present invention is the above-described yeast production method, wherein the yeast is Saccharomyces cerevisiae. A preferred embodiment of the present invention is the above-described yeast production method, wherein the yeast is a Schizosaccharomyces pombe. As demonstrated by the Examples, by controlling the proofreading function of DNA polymerase in a yeast gene disruption strain (especially the och1 gene disruption strain, etc.) Yeast that can be produced with high efficiency can be produced.

  In a preferred embodiment of the present invention, the yeast gene disruption strain is a strain having one or more disruptions selected from the group consisting of {och1 disruption, mnn1 disruption, mnn4 disruption, and alg3 disruption}, or The yeast production method according to any one of the above, which is a strain having one or two or more mutations selected from the group consisting of {och1 mutation, mnn1 mutation, mnn4 mutation, and alg3 mutation}. That is, as demonstrated by the examples, when a gene-disrupted strain in which a gene such as och1 related to sugar chain elongation is disrupted is used, high-temperature sensitivity is avoided while the ability to produce mammalian proteins is exhibited, or the growth is restored. Yeast that produces β-glucan with high efficiency can be obtained.

  In a preferred embodiment of the present invention, the step of controlling the proofreading function of the DNA polymerase includes the step of adjusting the error prone frequency of the DNA polymerase in a gene function deficient strain such as a gene disruption strain or a gene mutant of yeast. And, more specifically, a step of modifying the amino acid sequence of Pol3 (for example, the polypeptide described in SEQ ID NO: 1) in a gene function-deficient strain such as a gene disrupted strain of budding yeast. It is the manufacturing method of the yeast in any one of the said containing. A preferred embodiment of the present invention includes any of the above steps comprising a step of modifying the amino acid sequence of Cdc6 (for example, the polypeptide described in SEQ ID NO: 7) in a gene function-deficient strain such as a gene disrupted strain of fission yeast. This is a method for producing yeast.

  In a preferred embodiment of the present invention, as demonstrated by Examples, the yeast gene disruption strain is a budding yeast gene disruption strain into which och1 disruption, mnn1 disruption, and mnn4 disruption are introduced, and the DNA polymerase proofreading In the step of controlling the function, a DNA in which the 962nd base in the POL3 gene represented by SEQ ID NO: 2 is substituted from A to C and the 968th base is substituted from A to C is added to the yeast gene-disrupted strain. The method for producing yeast according to any one of the above, which comprises a step of transforming. About this aspect, since it demonstrated using budding yeast by the Example mentioned later, it is thought that it can be used about yeast in general.

  In another preferred embodiment of the present invention, as demonstrated by the examples, the yeast gene disruption strain is a fission yeast gene disruption strain into which och1 disruption has been introduced, and controls the calibration function of the DNA polymerase. Any of the above steps comprising transforming the yeast gene-disrupted strain with DNA in which the 898th to 906th bases GAT ATT GAA in the cdc6 + gene represented by SEQ ID NO: 8 are replaced with GCC GGC GCT The present invention relates to a method for producing yeast as described above. The fission yeast gene disruption strain into which och1 disruption has been introduced may be one into which only och1 disruption has been introduced, for example, one selected from the group consisting of {mnn1 disruption, mnn4 disruption, and alg3 disruption} Alternatively, it may be a strain having two or more disruptions, or a strain having one or two or more mutations selected from the group consisting of {och1 mutation, mnn1 mutation, mnn4 mutation, and alg3 mutation}.

  A preferred embodiment of the present invention is the yeast production method according to any one of the above, wherein the yeast that has avoided high-temperature sensitivity or restored growth is a budding yeast or a fission yeast that produces a glycoprotein having a mammalian sugar chain. About.

  A preferred utilization mode of the present invention is to produce a glycoprotein, wherein the yeast obtained by any one of the yeast production methods described above is cultured in a medium to produce a glycoprotein, and the glycoprotein is collected from the culture. Regarding the method. The glycoprotein obtained by using such a production method is, for example, a glycoprotein having a mammalian sugar chain, and can be effectively used for medicines and the like. As yeast in this utilization mode, budding yeast or fission yeast can be mentioned. Similarly, in a preferred embodiment of the present invention, yeast obtained by any one of the yeast production methods described above is cultured in a medium to produce β-glucan, and the β-glucan is collected from the culture. The present invention relates to a method for producing β-glucan. The β-glucan obtained by using such a production method can be effectively used, for example, for various functional foods and medicines. As yeast in this utilization mode, budding yeast or fission yeast can be mentioned.

  The second aspect of the present invention is the production of a yeast that avoids high-temperature sensitivity or restores growth, comprising a step of controlling the proofreading function of DNA polymerase in a gene function-deficient strain such as a gene disrupted strain or gene mutant of yeast. The present invention relates to a yeast obtained by the method, and a yeast obtained by a method for producing a yeast that produces β-glucan with high efficiency, comprising the step of controlling the proofreading function of DNA polymerase in a yeast gene function-deficient strain. This yeast avoids high-temperature sensitivity or restores growth and produces β-glucan with high efficiency. Even though it is a gene function-deficient strain such as a gene-disrupted strain, Have. As yeast in this utilization mode, budding yeast or fission yeast can be mentioned.

As a specific yeast, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (〒305-8565)
Yeast 1-1-1, Tsukuba Center, Tsukuba City, Ibaraki Prefecture, Central 6) includes yeasts deposited under the accession number “FERM P-20955”, “FERM P-20956” or “FERM P-21145”. These yeasts are those that have avoided high temperature sensitivity or have recovered their growth potential, and if these yeasts are used, proteins similar to mammals can be produced. Moreover, these yeasts produce β-glucan with high efficiency. That is, the present invention provides a saccharide using a yeast produced by any of the methods described above, or a yeast deposited as “FERM P-20955”, “FERM P-20956” or “FERM P-21145”. A method for producing a protein or a method for producing β-glucan is also provided.

  The third aspect of the present invention relates to a yeast produced by any one of the above-described yeast production methods, a glycoprotein production method using any one of the above yeasts, or a β-glucan production method. As demonstrated by the examples, these yeasts have excellent glycoprotein production ability and excellent β-glucan production ability. Therefore, by using these yeasts, glycoproteins or β-glucan can be effectively used. Can be manufactured.

  The fourth aspect of the present invention relates to a method for producing yeast having the ability to express foreign genes, comprising the step of controlling the proofreading function of DNA polymerase in a gene function deficient strain such as a gene disrupted strain or gene mutant of yeast. That is, if a foreign gene such as a human-derived gene can be expressed using yeast and a foreign protein can be obtained, it is effective for the development of pharmaceuticals and the like. Yeast having expression ability can also be produced. The invention according to this aspect can employ various usage modes similar to those according to the first aspect. As yeast in this utilization mode, budding yeast or fission yeast can be mentioned.

  According to the present invention, a method for producing a gene function-deficient strain such as a yeast gene-disrupted strain (or gene mutant strain) that avoids high-temperature sensitivity or has recovered its growth property, and a yeast obtained by such a production method are provided. be able to.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel mutant strain of yeast that recovers growth delay while maintaining the ability to produce glycoproteins having mammalian sugar chains and is excellent in high temperature resistance and protein production ability, and such a mutant strain are produced. Can provide a way to do.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the glycoprotein which has a mammalian type sugar chain using the yeast manufacturing method which avoided the above high temperature sensitivities or recovered the growth property can be provided.

  ADVANTAGE OF THE INVENTION According to this invention, the yeast obtained by the manufacturing method of the yeast gene disruption strain (or gene mutant strain) which produces (beta) -glucan highly efficiently, and such a manufacturing method can be provided.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of (beta) -glucan using the manufacturing method of the yeast which produces the above beta-glucans with high efficiency can be provided.

  According to the present invention, it is possible to provide a method for producing a yeast having the ability to express a foreign gene, and a method for producing a foreign protein using such a production method.

  The first aspect of the present invention is a yeast that avoids high-temperature sensitivity or restores growth properties, comprising a step of controlling a proofreading function of DNA polymerase using a gene function-deficient strain such as a gene disruption strain or gene mutant strain of yeast. And a method for producing yeast that produces β-glucan with high efficiency. And the 2nd side surface of this invention is related with the yeast manufactured according to the manufacturing method. Furthermore, a preferred embodiment of the present invention is a method for producing a glycoprotein having a mammalian sugar chain, or β-glucan, including such a method for producing yeast. In the present specification, the “gene function deficient strain” means a strain into which either or both of gene disruption and gene mutation have been introduced. A strain into which both gene disruption and gene mutation have been introduced is also included in the gene disruption strain or gene mutation strain.

  The yeast used in the present invention is not particularly limited as long as it is generally called yeast, and budding yeast, fission yeast, and the like can be appropriately used. Representative yeasts include those belonging to the family Saccharomycesaceae or Schizosaccharomycesaceae. As a more specific yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae), which is a kind of budding yeast, and Schizosaccharomyces pombe (schizosaccharomyces pombe), which is a kind of budding yeast, are widely used. can give. Examples of other yeast that can be used in the present invention include black yeast (Aureobasidium pullulans). Among these, the budding yeast and fission yeast demonstrated in the examples are preferable, but the present invention is not particularly limited to the budding yeast and fission yeast, and can be widely applied to all yeasts. In particular, for fission yeast, it is known that, like budding yeast, disruption of a predetermined gene can effectively prevent the addition of mannose to a sugar chain and produce a glycoprotein having a mammalian sugar chain. (Takehiko Yoko-o et al., FEBS Letters 489 (2001) 75-80; Clinton E. Ballow et al., Proc. Natl. Acad. Sci. USA Vol. 91. pp9327-Tak. et al., Biochemical and Biophysical Research Communications 330 (2005) 813-820; Sandra Fanchitti et al., Journal. of Cell Biology, Vol. 143, No. 3, 1988, pp 625-636). As for Pichia pastoris, see, for example, Water Verken et al. , Applied and Environmental Microbiology, Vol. 70, no. 5, 2004, pp 2639-2646, and yeast Yarrowia Lipolytica is described in, for example, Stepney Barney-Verdier et al. , Microbiology (2004), 150, p2185-2195. In the present invention, the genes involved in the proofreading function of the DNA polymerase that is modified to introduce mutations are considered to be not strongly related to the sugar chain-related genes, so the genes disclosed in these documents By adjusting the proofreading function of the DNA polymerase for the disrupted strains, or gene disrupted strains that can be easily obtained by those skilled in the art from these gene disrupted strains, avoiding high temperature sensitivity as in the examples of the present invention or It is believed that yeast that has recovered its growth ability can be obtained.

  The yeast gene-disrupted strain used in the present invention is not particularly limited as long as it is a strain in which some gene in wild-type yeast is disrupted. A strain in which some gene has been disrupted generally has a lower resistance to high temperatures and higher growth than wild-type yeast. ADVANTAGE OF THE INVENTION According to this invention, the strain | stump | stock which introduce | transduced the mutation by controlling the proofreading function of DNA polymerase and recovered the function reduced by gene disruption can be provided. Furthermore, by introducing a mutation by controlling the proofreading function of DNA polymerase, it is possible to exert a function that is not found in wild strains or is poor in wild strains. As described later, in the examples, it was shown that high temperature sensitivity was avoided by using a triple disruption strain of Saccharomyces cerevisiae and a triple disruption strain of fission yeast, and proliferative properties were restored. It is not limited to budding yeast or fission yeast triple disruption strains, and can be used for various yeast gene disruption strains. Hereinafter, specific gene-disrupted strains will be described.

  Japanese Patent No. 3091851 discloses a double disruption strain (Δoch1 mnn1) having an mnn1 disruption in the OCH1 gene disruption strain (Δoch1) (for example, see Example 1 of the publication). That is, when the yeast gene disruption strain used in the present invention is a double disruption strain (Δoch1 mnn1), such a double disruption strain may be obtained according to the method described in the publication. According to the publication, if such a double disruption strain is used, a large amount of a high-mannose glycoprotein having the same core type sugar chain as that of high mannose produced by mammalian cells such as humans or this sugar chain structure is produced. And it is said that it can be produced with high purity. In the yeast production method of the present invention, if this double disruption strain is used, high temperature sensitivity is avoided and proliferative properties are restored, so that the same core type sugar chain as high mannose produced by mammalian cells is effectively obtained. And so on. The publication also discloses disrupted strains (Δoch1 mnn1 his1 and / or his3), and the yeast gene disrupted strains of the present invention may contain them.

  Japanese Patent No. 3091851 discloses a triple disruption strain (Δoch1 mnn1 mnn6) having och1 destruction, mnn1 destruction, and mnn6 destruction (for example, see Example 1 of the publication). That is, when the yeast gene disruption strain used in the present invention is a triple disruption strain (Δoch1 mnn1 mnn6) or the like, these strains may be obtained according to the method described in the publication. And according to the same publication, by using such a triple disruption strain or the like, a high mannose glycoprotein having the same core type sugar chain as that of a high mannose produced by mammalian cells such as humans or this sugar chain structure can be obtained. It is said that it can be produced in large quantities and with high purity. In the yeast production method of the present invention, if these strains are used, high temperature sensitivity is avoided and proliferative properties are restored, so that the same core type sugar chain as high mannose produced by mammalian cells can be effectively obtained. It can be produced. The publication also discloses disrupted strains (Δoch1, mnn1, mnn6, his1 and / or his3, ura3) and the like, and the yeast gene disrupted strains of the present invention may contain them. Furthermore, according to the technique disclosed in the publication, a double disrupted strain in which the OCH1 gene and the MNN1 gene are disrupted can be obtained.

  JP-A-9-266792 discloses a quadruple disruption strain in which the functions of the MNN4 gene and the KRE2 gene are disrupted in addition to the Δoch1 Δmnn1 disruption. As disclosed in the publication, for example, a sporulation medium in which mutant strains and gene-disrupted strains having different mating types are joined and the diploid cells produced are deficient in a nitrogen source [for example, F. Sherman, Methods in Enzymology, vol. 194, p. 17 (1991)], various varieties can be generated by meiosis, separating the four spores generated thereby individually under a microscope, and examining their phenotypes.

  International Publication WO 01/014522 (Patent Document 1) discloses a triple disruption strain having och1 disruption (Δoch1), mnn1 disruption (Δmnn1) and mnn4 disruption (Δmnn4). That is, among genes involved in the biosynthesis of an outer sugar chain specific to yeast, a gene (OCH1) encoding α-1,6 mannosyltransferase that performs the first extended addition reaction, mannose at the non-reducing end of the sugar chain Disclosed is a gene-disrupted yeast strain in which the function of the gene (MNN1) encoding α-1,3 mannosyltransferase that adds mannose and the gene (MNN4) that controls addition of mannose-1-phosphate is disrupted. TIY20 used in the examples of the present invention has a mat different from that of TIY19 disclosed in the literature, and is obtained from the same clone by four-molecule analysis. Tetramolecular analysis can be performed, for example, according to a method described in Dan Burke et al. (Translated by Junichi Ohya et al.), Yeast Genetic Experiment Manual, published on Mar. 10, 2002.

  International Publication WO01 / 014522 (Patent Document 1) discloses a budding yeast disruption strain into which disruption including och1 disruption, mnn1 disruption, mnn4 disruption and alg3 disruption is introduced, as well as OCH1 gene, MNN1 gene, and MNN4 gene And a budding yeast disruption strain in which the ALG3 gene is disrupted. In addition, in this publication, in addition to the mutant traits resulting from the gene disruption, an auxotrophic mutant trait selected from the group consisting of ura3 mutation, his3 mutation, leu3 mutation, leu2 mutation, ade2 mutation, trp1 mutation, and can1 mutation. A yeast mutant strain having According to the publication, these mutant strains can easily introduce foreign genes using auxotrophic selection markers, and by using these mutant strains, mammalian sugar chains or mammalian sugars and other sugars can be introduced. It is disclosed that a glycoprotein having a chain can be produced in a large amount and with high purity. Therefore, in the method for producing yeast of the present invention, if the mutant strain disclosed in the publication is used, high-temperature sensitivity is avoided and proliferative properties are restored, so that mammalian glycoproteins and the like can be produced effectively. Conceivable. Furthermore, it is considered that the mutant strain in which the high temperature sensitivity is avoided and the growth ability is restored in this way produces β-glucan with high efficiency.

  Thus, for example, as a gene function-deficient strain such as a yeast gene-disrupted strain or gene mutant strain, it has one or more disruptions selected from the group consisting of {och1 disruption, mnn1 disruption, mnn4 disruption, and alg3 disruption}. And a strain having one or more mutations selected from the group consisting of {och1 mutation, mnn1 mutation, mnn4 mutation, and arg3 mutation}. In other words, not only gene disruption strains due to gene disruption due to specific gene function deficiencies, but also gene mutation strains due to gene mutations have the same problems as described above. By controlling the calibration function of DNA polymerase, It is possible to obtain a yeast that avoids high temperature sensitivity or restores the growth ability, and thereby β-glucan can be produced with high efficiency. Among gene-deficient strains such as gene-disrupted strains or gene-mutated strains, yeast gene-disrupted strains having only och1 disruption or och1 disruption and other mutations can be preferably used. JP 2001-161376 discloses a fission yeast och1 disruption strain (Δoch1) that has lost the function of the glycosyltransferase gene och1 + of fission yeast. In the present invention, an och1 disruption strain (Δoch1) of fission yeast may be used as a gene disruption strain of yeast. Therefore, for example, even when fission yeast is used as the yeast, a gene-disrupted strain having only och1 disruption or och1 disruption and other mutations can be preferably used.

  The step of controlling the proofreading function of DNA polymerase As the step of controlling the proofreading function of DNA polymerase in a gene function deficient strain such as a gene disruption strain or gene mutant of yeast, the error prone frequency of DNA polymerase in a gene function deficient strain of yeast is determined. Examples include a step that regulates, and more specifically, those that include a step of modifying the amino acid sequence of Pol3 in a gene function-deficient strain such as a gene disrupted strain of Saccharomyces cerevisiae. Also included are those that include a step of modifying the amino acid sequence of Cdc6 in a gene function-deficient strain such as a gene disrupted strain of fission yeast.

  As used herein, “error-prone frequency” refers to the level of nature of error-prone. The error prone frequency can be expressed by, for example, the absolute number of mutations in the gene sequence (the number of mutations themselves) or the relative number (ratio of the number of mutations in the full length). Alternatively, when referring to an organism or enzyme, the error-prone frequency may be expressed as the absolute or relative number of mutations in the gene sequence per reproduction or division of an organism. Unless otherwise stated, the error-prone frequency is expressed as the number of errors per replication process in a gene sequence. Error-prone frequency is sometimes referred to herein as “accuracy” as an inverse measure. The error-prone frequency being uniform means that the error-prone frequencies are substantially equal to each other when referring to factors (polymerase or the like) responsible for the replication of a plurality of genes. On the other hand, when the error-prone frequency is non-uniform, it means that a significant difference exists in a factor (polymerase or the like) responsible for the replication of a plurality of genes.

  In this specification, “adjusting the error-prone frequency” means changing the error-prone frequency. Such adjustment of error-prone frequency includes increasing and decreasing error-prone frequency, but preferably increases error-prone frequency. Examples of methods for adjusting the error-prone frequency include modification of a DNA polymerase having a proofreading function, insertion of a factor that inhibits or suppresses a polymerization reaction or extension reaction during replication, and a factor that promotes these reactions Inhibition, suppression, single or multiple base deficiency, DNA repair enzyme deficiency, removal of abnormal base, modification of enzyme with repair factor function, mismatch base pair repair factor modification, reduction of accuracy of replication itself, etc. Is not limited to them. The adjustment of the error-prone frequency may be performed on both DNA double strands or only on one side. Those performed on only one of the DNA double strands are preferred because harmful mutagenesis is reduced.

As used herein, “error prone” refers to a property that is prone to error in replication of a gene (DNA or the like) (that is, replication error). Error prone is mainly affected by the accuracy of the calibration function of an enzyme having a calibration function (for example, a DNA polymerase). As used herein, “replication error” refers to an error in nucleotide incorporation that occurs in the process of gene (DNA, etc.) replication. In general, the frequency of replication errors is extremely low in a living body, about once every 10 ⁸ to 10 ¹² times. The reason for the low frequency of replication errors is that replication occurs when nucleotide incorporation forms a complementary base pair with the nucleotide into which the template DNA is incorporated, and the proofreading function of an enzyme such as DNA polymerase, ie 3 ′ → 5 'Exonuclease has a function to detect and immediately cut out a nucleotide that is not complementary to the template when it is mistakenly incorporated. Therefore, in the present invention, the frequency of error prone in replication can be adjusted by the failure of specific base pairing, the failure of the calibration function, or the like.

  As used herein, “error-free” refers to the property that there is almost no error in replication of a gene (DNA or the like), preferably substantially no error. Error free is mainly affected by the accuracy of the calibration function of an enzyme having a calibration function. As used herein, error-prone and error-free can be classified absolutely (ie, determined by the level of error-prone frequency, etc.) or relative (factors responsible for the replication of two or more genes (eg, , DNA polymerase, etc.) can be classified as error prone with a higher error prone frequency and error free with a lower error prone frequency.

In this specification, “DNA polymerase” refers to an enzyme having a function of polymerizing DNA by liberating pyrophosphate from four types of deoxyribonucleoside 5′-triphosphates. This includes Cdc6. For the DNA polymerase reaction, template DNA, primer molecules, Mg ^{2+ and the} like are required. A nucleotide complementary to the template is sequentially added to the 3′-OH end of the primer to extend the molecular chain.

  In the present invention, in order to control the proofreading function of DNA polymerase, yeast cells may be converted into an unbalanced mutator by some method. In order to make a yeast cell an unbalanced mutator, a gene region involved in the proofreading function of a DNA polymerase gene may be destroyed or some mutation may be introduced into such a gene region according to a known method. Regarding gene disruption and gene mutation introduction methods, for example, gene regions involved in the proofreading function of DNA polymerase genes are known, and thus the base sequences of these gene regions are, for example, 1 to 100, preferably 1 It may be transformed by substituting with 10 to 10, more preferably 1 to 3 with other bases. For example, by expressing in a target yeast cell a mutant protein that has lost the proofreading function of polymerase δ (Polδ), which is believed to be involved in replication of the lagging strand during eukaryotic chromosomal DNA replication, It can be made into a tater. That is, as a method for expressing a Polδ mutant protein in a target yeast cell, an artificially obtained Polδ mutant gene is transformed into a target yeast to cause the mutant gene to be expressed and function. can give. That is, as a method for creating a proofreading function loss mutation type of Pol3 which is Polδ in budding yeast, a part of the amino acid sequence of the proofreading function active site is artificially substituted using a previously cloned wild-type POL3 gene as a template, The calibration function may be controlled. In addition, a method of cloning and using the gene from a strain having the same trait as that of the pol3 mutant and already identified as a natural mutant may be used (in the examples described later, the latter method is used). Using.). The DNA polymerase gene may include not only a structural gene of DNA polymerase but also both transcriptional and / or translational regulatory sequences such as a promoter of DNA polymerase.

  As another example, a mutant protein in which the proofreading function of Cdc6 (Cell division cycle 6), which is Polδ in fission yeast, is lost is expressed in the target yeast cell, thereby making the fission yeast cell an unbalanced mutator. can do. That is, as a method for expressing Cdc6 mutant protein in a target yeast cell, artificially obtaining a cdc6-mutated gene and transforming the target yeast to express the mutant gene and make it function Can also be raised. Specifically, the proofreading function loss type cdc6 (cdc6-) can be obtained by the same production method as the pol3 mutant gene.

  In Examples described later, the pol3-01 mutant gene (base sequence shown by SEQ ID NO: 4) in the POL3 gene consisting of the base sequence shown by SEQ ID NO: 2 is the 962nd base in the POL3 gene shown by SEQ ID NO: 2. Is substituted from A to C, and the 968th base is substituted from A to C. However, the method for controlling the proofreading function of DNA polymerase in Saccharomyces cerevisiae is not limited to the above method. For example, 1 to 10 nucleotide sequences at different positions including substitution at the above positions ( Various methods such as a method of substituting 2 to 5 bases, more preferably 2 to 5 bases of the 898th to 980th positions of the POL3 gene represented by SEQ ID NO: 2 are included. Further, the cdc6-1 mutant gene (base sequence shown by SEQ ID NO: 10) in the cdc6 + gene consisting of the base sequence shown by SEQ ID NO: 8 is the 898th to 906th base GAT ATT in the cdc6 + gene shown by SEQ ID NO: 8. GAA is replaced with GCC GGC GCT. However, the method for controlling the proofreading function of DNA polymerase in fission yeast is not limited to the above method. For example, 1 to 10 nucleotide sequences at different positions including substitution at the above position ( Various methods are included, such as a method of preferably replacing 2 to 5 and more preferably 2 to 5 bases from 898th to 980th of the cdc6 + gene represented by SEQ ID NO: 8.

  In this specification, when the proofreading function is “lower than that of the wild type”, when referring to an enzyme or the like having a certain proofreading function, the proofreading function of the enzyme is lower than that of the wild type (that is, The number of mutations remaining after the calibration process is larger than the number of mutations remaining after the wild type calibration process). Comparison with such wild type can be done by relative or absolute representation. Such a comparison can also be made, such as by error-prone frequency.

  As used herein, “mutation” refers to a state of a gene (nucleic acid or amino acid) sequence that causes a change in the sequence of the gene or a gene caused by the change. As used herein, for example, mutations are used for gene sequence changes that occur for proofreading functions. In this specification, unless otherwise stated, mutation is used synonymously with modification.

  It is most common to perform mutagenesis in an organism to create useful mutants. The mutation usually means a change in the base sequence encoding the gene, and includes a change in the DNA sequence. Mutations can be broadly divided into the following three types according to the effects on the individuals in which they occur: A) Neutral mutations: This mutation applies to most mutations and has almost no effect on the growth and metabolism of organisms. There is no. B) deleterious mutation: This mutation is less frequent than the neutral mutation. Inhibit the growth or metabolism of an organism. The harmful mutation includes a lethal mutation that destroys a gene essential for growth. In the case of microorganisms, although it varies depending on the species, the ratio of harmful mutations to all mutations is usually about 1/10 to 1/100. C) Beneficial mutation: This mutation is beneficial for the breeding of organisms. Its frequency of occurrence is very low compared to neutral mutations. Therefore, in order to obtain an organism individual into which a beneficial mutation has been introduced, a large organism population and a long time are required. In addition, a sufficient effect of breeding an organism rarely appears with only a single mutation, and it is often necessary to accumulate a plurality of beneficial mutations. The introduction of these mutations can be performed, for example, according to the method described in Dan Burke et al. (Translated by Junichi Ohya et al.), Yeast Genetic Experiment Manual, published on Mar. 10, 2002.

  Below, the case where POL3 gene is made into object as a process which controls the proofreading function of DNA polymerase and makes a yeast cell an imbalance mutator is demonstrated. In addition, when the cdc6 gene or the like in fission yeast is targeted as a DNA polymerase that controls the proofreading function, the yeast cell can be converted into an unbalanced mutator as in the case of targeting the POL3 gene. Pol3 and Cdc6 are DNA replication enzymes collectively called polymerase δ (Polδ) in eukaryotes, and are genes corresponding to Polδ in budding yeast and fission yeast, respectively. Specifically, the POL3 gene is saccharomyces cerevisiae W303-1A (ura3, leu2, his3, trp1, ade2) [Kainuma et al., Glycobiology, Vol. 9, 133-141 (1999)] can be obtained by PCR using the genome obtained from the strain as a template. A primer provided with a restriction enzyme cleavage portion may be used so that a portion encoding the Pol3 protein can be easily cut out. More specifically, after cloning the budding yeast POL3 gene into an appropriate vector, a primer can be designed to introduce a mutation so as to control the proofreading function of Pol3, thereby obtaining mutant pol3.

  In order to express the mutant pol3 in yeast, an expression cassette is constructed by inserting a promoter upstream and a terminator downstream, and introducing this into an expression vector. If a promoter and terminator are already present in the expression vector into which the gene is to be introduced, only the fusion gene may be introduced between them using the promoter and terminator without constructing an expression cassette.

  The promoter in the expression cassette is not particularly limited as long as it is a promoter that is generally used in a yeast expression system and can express a fusion gene introduced in a transformed yeast. For example, PGK, GAP, TPI, GAL1 , GAL10, ADH2, PHO5, and CUP1, among which GAP promoter is preferable.

  On the other hand, the terminator is generally used in yeast expression systems, and any terminator may be used as long as it is present downstream of the introduced fusion gene and can terminate transcription. Examples thereof include ADH1, TDH1, TFF, and TRP5.

  The expression vector for introducing the expression cassette is not particularly limited as long as it is generally used in yeast expression systems. As specific expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, and pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, and pC194), yeast-derived plasmids (eg, pSH19, and pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus and vaccinia virus, and insect pathogenic viruses such as baculovirus can be used, and yeast-derived plasmids can be preferably used.

  The plasmid used for yeast transformation is not particularly limited as long as it is a plasmid that can be used for yeast transformation, for example, yeast episome plasmid abbreviated as YEp, abbreviated as YRp. Yeast self-replicating plasmid (yeast replicating plasmid) and the like. The yeast episomal plasmid vector is a vector that contains the 2μ plasmid sequence inherent in yeast and can be replicated in host yeast cells using its origin of replication. The yeast episomal expression vector used in the present invention preferably contains at least the ARS sequence of the yeast 2μ plasmid sequence and can be propagated extrachromosomally in the host yeast. Specific examples of the plasmid include YEp51, pYES2, YEp351, YEp352, and pREP.

  The yeast episomal expression vector is preferably a shuttle vector that can be propagated inside the Escherichia coli so that subcloning can be performed in recombinant Escherichia coli, and more preferably includes a selection marker gene such as an ampicillin resistance gene. The expression vector also contains a marker gene that allows selection of yeast clones by auxotrophy or drug resistance when recombinant yeast is produced. Examples of the marker gene include HIS3, TRP1, LEU2, URA3, ADE2, CAN1, SUC2, LYS2, and CUP1 (edited by Taiji Oshima, Biochemical Experimental Method 39, Yeast Molecular Genetics Experimental Method, 119-144). (1996)). These are merely examples, and may be appropriately selected according to the genotype of the yeast strain used as the host for gene transfer. A series of techniques relating to the construction of the above-described fusion gene expression plasmid can be appropriately carried out by those skilled in the art with reference to the description in Examples below or by conventional techniques.

  A promoter, enhancer, splicing signal, poly A addition signal, selection marker, SV40 replication origin, DNA encoding the tag, and the like may be added to the expression vector. The expression vector may be a fusion protein expression vector. Commercially available fusion protein expression vectors include pGEX series (Amersham Pharmacia Biotech), pET CBD Fusion System 34b-38b (Novagen), pET Dsb Fusion Systems 39b and 40b (Novagen), and pET GST Fst 41 42 (Novagen).

  In the present invention, examples of host yeast transformed with the mutant pol3 gene expression vector (mutator) include those using yeasts belonging to the genus Saccharomyces and Candida, but are not particularly limited. Examples of Saccharomyces yeasts include Saccharomyces cerevisiae KK4 strain, Y334 strain, Inv-Sc1 strain, and W303 strain. In the present invention, host yeast transformed with the above mutant cdc6 gene expression vector includes those using yeast belonging to the genus Schizosaccharomyces or Candida, but is not particularly limited. Examples of yeast belonging to the genus Schizosaccharomyces include Schizosaccharomyces pombe and TN8 strain.

  In order to transform yeast with a fusion gene expression vector, for example, the following methods can be mentioned. A known method such as a method of incubating with lithium phosphate, adding DNA and PEG and incubating, or electroporation can be used (Becker and Guarente, Methods Enzymol., 194, 182-187 (1991)). In addition, spheroplast cells in which cell walls are digested with an enzyme are incubated with PEG and DNA in the presence of calcium ions derived from calcium dichloride or the like (Hinen et al., Proc. Natl. Acad. Sci. USA, 75: 1929 (1978)) or a method of transformation by irradiating cells with DNA-attached particles (Fox TD et al., 1988. Plasmids can stably transform). yeast mitochondria racking endogeneous mtDNA.Proc.Nat.Acad.Sci.85: 7288-7292) may be used.

  A yeast transformation method (Japanese Patent No. 3682530) comprising a step of maintaining a logarithmically growing yeast cell in a solution containing a gene to be introduced into the yeast cell and polyethylene glycol may be used.

  An appropriate selection marker is used for screening for transformed yeast. As an example, it is desirable to use a gene involved in metabolism on the chromosomal DNA of the host cell. That is, by using a host cell that does not function the above gene on chromosomal DNA by appropriate means such as mutation, and transforming an expression vector containing the corresponding normal gene, transformation containing a normal metabolic gene Those which can be screened by growing only cells are preferred. Specifically, a selection marker gene that is widely used such as URA3 and LEU2 as described above is connected to an expression vector. In the case of the chromosomal integration type (YIp type), these genes also serve as screening markers.

  In order to introduce many mutations into transformed transformed yeast, the cells are cultured so that division can be repeated over several generations. Specifically, it is cultured overnight in 5 ml of medium, repeatedly scaled up to 2 ml, 50 ml, and 100 ml. The culture is repeated in a state where yeast can divide for a period of about one week. The method for culturing the transformed yeast can be performed according to the usual method used for culturing yeast. As the medium, a medium containing a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by yeast and capable of efficiently cultivating the transformant may be used. Specifically, a YPD medium, a YPG medium, a YPDG medium YPAD medium, glucose synthesis minimum medium (SD), iodine-added minimum medium (SMM), Hartwell complete medium (HC), GAL fermentation test medium, sporulation medium, or the like can be used as appropriate. In addition, for example, a synthetic medium (carbon source, nitrogen source, inorganic salt, amino acid) added with various medium components supplied from Difco and excluding amino acids that can be supplied by markers required for plasmid replication / retention (Including vitamins) and the like (Sherman, Methods Enzymol., 194, 3-57 (1991)).

  It is appropriate to adjust the pH of the medium to 6-8. The pH may be adjusted by controlling the amount of inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia or the like added. The culture may be performed at 28 to 32 ° C., preferably 30 ° C., for about one week (for example, 1 day to 1 month, preferably 5 days to 10 days) according to a conventional method while appropriately adding aeration and stirring. In particular, in order to culture the TIY20 strain efficiently, it is desirable to add KCl and sorbitol and culture at 30 ° C. However, in order to apply a gentle selective pressure, the culture may be performed in a medium to which KCl and sorbitol are not added. . Further, in order to obtain a gentle selection pressure, the culture may be performed at a temperature slightly higher than 30 ° C. such as 31 ° C. to 35 ° C. (or 32 ° C. to 33 ° C.).

  Yeast that avoids high-temperature sensitivity or restores growth ability Yeast that avoids high-temperature sensitivity or restores growth ability means yeast produced by the above-described production method. Specific examples include budding yeast or fission yeast that produce glycoproteins having mammalian sugar chains. More specifically, a yeast obtained by modifying a gene relating to a proofreading mechanism of a DNA polymerase in a known gene-disrupted strain or a gene-disrupted strain that can be produced from a known gene-disrupted strain according to a known method is exemplified. .

  Glycoprotein having a mammalian type sugar chain The glycoprotein having a mammalian type sugar chain may be produced by the above-mentioned known gene disruption strains, and a method for isolating and purifying glycoproteins from gene disruption strains, A method disclosed in any document described in this specification or a known method may be appropriately used. For example, after the culture is completed, the cells are collected by centrifugation and suspended in an aqueous buffer solution. Thereafter, the cells are crushed using an ultrasonic crusher, a French press, a homogenizer, a dynomill or the like as appropriate to obtain a cell-free extract. The cell-free extract thus obtained is centrifuged to obtain a supernatant. From the supernatant, a solvent extraction method, a salting-out method using ammonium sulfate, a precipitation method using an organic solvent, a resin such as diethylaminoethyl-sepharose, etc. What is necessary is just to combine the anion exchange chromatography method using this, an affinity chromatography method, etc. suitably.

  In the present specification, Man represents mannose, and GlcNAc represents N-acetylglucosamine. An asterisk (*) indicates a site capable of phosphorylation. Specific examples of glycoproteins having mammalian sugar chains include glycoproteins having an oligosaccharide chain represented by the following formula (I) or (II) as an asparagine-linked sugar chain.

  Yeasts that produce β-glucan with high efficiency Yeasts that produce β-glucan with high efficiency include yeasts produced by the production method described above, as well as yeasts that have avoided high-temperature sensitivity or have recovered their growth properties. On the other hand, β-glucan can be obtained with high efficiency by using yeast that has been obtained without controlling the proofreading function of DNA polymerase in the yeast gene-disrupted strain and that has avoided high-temperature sensitivity or has recovered its growth properties. It is thought that it is possible. Examples of yeasts that produce β-glucan with high efficiency include budding yeast or fission yeast that produce β-glucan with high efficiency. More specifically, a yeast obtained by modifying a gene relating to a proofreading mechanism of a DNA polymerase in a known gene-disrupted strain or a gene-disrupted strain that can be produced from a known gene-disrupted strain according to a known method is exemplified. .

[beta] -glucan [beta] -glucan may be any one produced by the aforementioned known gene disruption strain. For example, specific β-glucan types include β-1,3-D-glucan and β-1,6 / 1,3-D glucan. As a method for isolating and purifying β-glucan from a gene-disrupted strain (particularly from a yeast cell wall), a known method can be appropriately used. For example, after the culture is completed, the obtained culture (cell) is collected by centrifugation and suspended in an aqueous buffer solution. Next, the cells are crushed using an ultrasonic crusher, a vortex mixer, a French press, a homogenizer, a dyno mill, or the like as appropriate to obtain a cell lysate. The obtained cell lysate is centrifuged to collect pellets (including cell walls) (resuspension, centrifugation, and pellet recovery are repeated an appropriate number of times). Then, after performing predetermined operations such as solvent removal, drying under reduced pressure, and resuspension, for example, a method of performing carbohydrate analysis using HPLC by the pyridylamino (PA) method is suitable. HPLC analysis by the method is more preferred.

  The second aspect of the present invention relates to a method for producing yeast having the ability to express foreign genes, comprising the step of controlling the proofreading function of DNA polymerase in a yeast gene disruption strain. The second aspect of the present invention is the same as the first aspect except that a gene disrupted strain into which a foreign gene has been introduced is used. A method for producing a gene-disrupted strain into which a foreign gene has been introduced may be appropriately performed according to a known method. The foreign gene in the second aspect of the present invention is not particularly limited as long as it is a gene derived from an organism other than the species, and examples thereof include human-derived human α-galactosidase A gene. As demonstrated by Example 5 described below, according to this method, a foreign gene can be effectively expressed. As in the first aspect, the second aspect of the present invention is not only a method for producing a yeast having the ability to express a foreign gene, but also a yeast having the ability to express a foreign gene produced by the production method, a foreign gene, Also provides protein and so on. The invention according to this aspect also comprises culturing yeast obtained by the above-described production method in a medium, expressing a foreign gene to produce a foreign protein, and collecting the foreign protein produced from the obtained culture. Thus, a method for effectively producing a foreign protein is also provided.

  Description of SEQ ID NOs: The sequences described in the sequence listing of the present specification are the following sequences. SEQ ID NO: 1 shows the amino acid sequence of Pol3. SEQ ID NO: 2 shows the base sequence of cDNA encoding POL3 gene. SEQ ID NO: 3 shows the amino acid sequence of pol3-01. SEQ ID NO: 4 shows the nucleotide sequence of cDNA encoding pol3-01 mutant gene. SEQ ID NO: 5 shows the base sequence of the forward primer used in the PCR reaction in Example 1. SEQ ID NO: 6 shows the base sequence of the reverse primer used in the PCR reaction in Example 1. SEQ ID NO: 7 shows the amino acid sequence of Cdc6. SEQ ID NO: 8 shows the nucleotide sequence of cDNA encoding cdc6 + gene. SEQ ID NO: 9 shows the amino acid sequence of cdc6-1. SEQ ID NO: 10 shows the base sequence of cDNA encoding the cdc6-1 mutant gene. SEQ ID NO: 11 shows the base sequence of the forward primer used in the PCR reaction in Example 6. SEQ ID NO: 12 shows the base sequence of the reverse primer used in the PCR reaction in Example 6. SEQ ID NO: 13 shows the base sequence of the forward primer used in the PCR reaction in Example 6. SEQ ID NO: 14 shows the base sequence of the reverse primer used in the PCR reaction of Example 6.

Description of deposited strain The YAB100 strain obtained in the examples described later has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of July 11, 2006, with the deposit number: FERM BP-11122 . . The YAB101 strain obtained in Examples described later has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of July 11, 2006 under the accession number: FERM BP-11123 .
The C2-11 strain obtained in the Examples described later has been deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on December 27, 2006 as the deposit number: FERM BP-11124 .

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

Production of Plasmid pAB100 Introducing Mutant Pol3 DNA Fragment The mutant pol3 DNA fragment was cloned into the SacI-SalI site of the multi-copy vector YEP352GAP2 for forced expression of budding yeast as follows, and plasmid pAB100 (see FIG. 1 (a)) was obtained. It was constructed. The amino acid sequence of Pol3 is shown in SEQ ID NO: 1, and the base sequence of DNA encoding Pol3 is shown in SEQ ID NO: 2. The pol3-01 mutant gene obtained in this example is the 962nd base in SEQ ID NO: 2. Is substituted from A to C, and the 968th base is substituted from A to C. That is, a mutation was introduced such that the amino acid residue encoded by the base sequence from the 961st to the 969th base sequence of SEQ ID NO: 2 was replaced from DIE to AIA. The amino acid sequence of pol3-01 thus obtained is shown in SEQ ID NO: 3, and the base sequence of the DNA encoding the pol3-01 mutant gene is shown in SEQ ID NO: 4. Specifically, the AMY128-1 strain (MATαpol3-01, ura3-52, leu2-1, lys1-1, ade2-1, his1-7, hom3-10, which is known as a natural mutator mutant, Using the genome of trp1-289) as a template, PCR reaction (forward primer: 5′-AGCTC GAGCTC (SacI) ATGAGTGAAAAAAGATCCCTTCCCATG-3 ′ (SEQ ID NO: 5), reverse primer: 5′- GCATTCGCGGCCGCC (NotI) TTACCTTTGCTTACAT '(SEQ ID NO: 6)), the pol3-01 mutant gene was amplified, and the amplified fragment was cloned into the SacI-NotI site of the pYES2 vector (the resulting plasmid was pY It was named ES2-pol3-01.) Further, the pol3-01 mutant gene is digested with restriction enzymes SacI and XhoI so that the pol3-01 mutant gene can be expressed under the control of the GAPDH promoter, and the SacI-SalI site of the YEp352GAP-II vector is digested. (The plasmid thus obtained was named pAB100).

Acquisition of high-temperature-resistant strain of sugar chain-modified strain Saccharomyces cerevisiae sugar chain-modified strain TIY20 (matα och1 :: hisG mnn1 :: hisG mnn4 :: hisG) is transformed with the plasmid pAB100 obtained as described above. Converted. TIY20 was obtained by four-molecule analysis from the same clone as TIY19 disclosed in WO 01/014522 (Patent Document 1). The resulting transformant (TIY20 / pAB100) was added to a budding yeast synthetic medium SD-U (6.7 g Yeast nitrogen base with amino acids (Difco laboratories), 20 g glucose, 0.77 g) to introduce more mutations. CMS-URA (Sunrise Science Products) (liquid) was cultured under controlled conditions so that it could divide as many as possible. Culturing was carried out for 3 days at 37 ° C., and the colonies that had grown were caught, and in order to remove pAB100 from the obtained strain, complete medium YPAD (10 g Yeast extract (Difco laboratories), 20 g peptone (Difco)) 0.2g adenine sulfate (Sigma), was streaked on 20gGlucose / 1L), and cultured to collect a single colony by 20. Were the colonies that can not grow in SD-U medium of these colonies.

Analysis of sugar chain length added to protein invertase in yeast 9 strains obtained in Example 1 (C15, C27, C28, C30, C3-20, C4-1, C3-3-1, C3-7-2) In order to examine the N-linked sugar chain length of C3-3-9), the N-linked sugar chain length added to the invertase produced in yeast was examined by the following method. Each strain was cultured in 5 ml of YPAD, and then cultured in 5 ml of YPSuc (10 g Yeast extract (Difcolaboratories), 20 g of peptone (Difco), 10 g of sucrose / 1 L) for 3 hours or more to recover the bacteria. The collected bacteria were 50 μl of SDS-PAGE sample buffer (15% Glycerol, 0.125M Tris-HCl (pH 6.8), 2 mM PMSF, 3% SDS, 0.1% Bromophenol blue, 1% 2-mercaptoethanol) and glass. Beads were added and vortexed. After centrifuging at 15,000 rpm for 5 minutes, 5 μl of each supernatant was electrophoresed on 5% SDS-PAGE (100 V, 3 hours). The gel was transferred to a reaction solution (3.4 g sucrose, 3 ml 3M Na-acetate / 100 ml), incubated at 37 ° C. for 30 minutes, and then washed twice with deionized water. Transferred to staining solution (2 g NaOH, 50 mg triphenyltetrazole chloride / 50 ml) and boiled until color developed. The result is shown in FIG. FIG. 2 is an SDS gel electrophoresis photograph replacing the drawing for examining the N-linked sugar chain length. The lanes in FIG. 2 show C15, C27, C28, C30, C3-20, C4-1, C3-3-1, C3-7-2, C3-3-9, TIY20, and W303-1A from the left. . From FIG. 2, it was found that the N-linked sugar chains added to the 9 strains of invertase obtained had the same sugar chain length as the parent strain TIY20.

Analysis of Growth Recovery Efficiency Next, the growth recovery efficiency of the strain obtained in Example 1 was analyzed. After culturing at 30 ° C. with 5 ml of YPAD, it was transferred to 10 ml of YPAD so that OD600 was 0.1, and cultured at 30 ° C. or 37 ° C. Bacteria were collected at each time point and examined for OD600. The result is shown in FIG. FIG. 3 is a graph instead of a drawing showing the growth recovery efficiency at 30 ° C. and 37 ° C. FIG. 3 shows that among the nine strains analyzed, C4-1 and C3-20 have higher growth rates at 30 ° C. than TIY20. FIG. 3 also shows that the growth ability of C4-1 and C3-20 was recovered, although TIY20 hardly grew at 37 ° C. The state of each strain is shown in FIG. 1 (b), and the fraction of FIG. 1 (b) is shown in FIG. 1 (c).

Sugar chain structure analysis The sugar chain structure of the mannoprotein in the strain obtained in Example 1 was analyzed. Bacteria cultured in 50 ml were collected, washed with water, suspended in 8 ml of 100 mM citrate buffer (pH 7.0), and autoclaved at 121 ° C. for 2 hours. The supernatant was collected by centrifugation, 24 ml of cold ethanol was added, and the mixture was allowed to stand at −20 ° C. for 30 minutes, and then centrifuged to collect a precipitate. After suspending in water, the protein solution was adjusted to 3 mg / ml, and treated with 5 μl Glycopeptidase F (4450 manufactured by Takara Bio Inc.). After incubation at 37 ° C. for 17 hours, water was added to 100 μl, phenol / chloroform / isoamyl alcohol (25/24/1) was added and stirred well, and the supernatant was collected by centrifugation (phenol chloroform extraction). Chloroform was added to the collected liquid, stirred, and then centrifuged to collect the supernatant (chloroform extraction) and dried up. The dried-up sample was pyridylaminated with a pyridylamine manual kit (4480 manufactured by Takara Bio Inc.) and then subjected to seven phenol chloroform extractions to remove excess reagents. After chloroform extraction, the supernatant was dried up and dissolved in water, followed by HPLC (Class-VP manufactured by Shimadzu Corporation, column, TOSOH TSK-GEL AMIDE-80 (φ4.6 mm × 250 mm), flow rate, 1 ml / min. , Detection, 320 nm (excitation), 400 nm (fluorescence), buffer A, acetonitrile, buffer B, 200 mM TEAA (elution of each saccharide by increasing the buffer concentration), buffer B gradient conditions, 0-40 minutes, 30 → 60% , 40-50 minutes, 30%). The result is shown in FIG. FIG. 4 is a graph showing the results of column analysis instead of a drawing for analyzing a sugar chain structure. In the figure, the number of mannose is indicated by M. The vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). FIG. 4 shows that various numbers of mannose are added in the wild strain W303-1A. On the other hand, from FIG. 4, in all of the obtained strains, a peak showing a sugar chain structure composed of the same 8 mannoses as the parent strain TIY20 was observed as a main peak. This indicates that the mutant strain in the example has a so-called mammalian sugar chain. That is, if the mutant strain in the examples is used, a protein having a desirable sugar chain can be obtained. That is, the present invention also provides a method for producing a protein using the mutant strain in the examples.

Chitinase analysis The secretion efficiency of the protein secreted from the strain obtained in Example 1 was analyzed. 40 mg wet chitin (Sigma) is added to the supernatant of the bacteria cultured in 40 ml and stirred overnight at 4 ° C. Centrifuge and collect chitin, then wash 3 times with PBS. After suspending in 100 μl SDS-PAGE sample buffer and treating at 100 ° C. for 10 minutes, 10 μl was subjected to SDS-PAGE and lectin blotted with ConA-biotin (Seikagaku Corporation). Detection was performed with Streptavidin-HRP (Seikagaku Corporation). Immobilon Western Chemiluminescent HRP Substrate (Milipore) was used as a detection reagent, and Fuji Film LAS1000 was used as a detection device. The result is shown in FIG. FIG. 5 is a graph replaced with a drawing for showing the secretion efficiency of the protein secreted from the mutant strain. FIG. 5 shows that the secretion efficiency of TIY20 is recovered in the obtained strain, although the secretion efficiency is about 50% of that of the wild type strain. In particular, in C4-1 and C3-20, the secretion efficiency recovered to the same level as the wild type strain. Therefore, the obtained strain C4-1 was deposited as YAB100 and C3-20 as YAB101, and the patent microorganism was deposited.

α-Galactosidase A activity measurement (verification of foreign gene expression ability) Vector (pRS4-GAP-αGalA) (Chiba, Y. et al., Glycobiology) having an expression cassette in which a human α-galactosidase A gene is linked downstream of the GAPDH promoter , 12, 821-828, 2002) were transformed into C4-1, C3-20, C3-7-2, and C27, respectively. The obtained transformant was cultured for the time shown in FIG. 6, and the culture solution (including bacteria) was used as an enzyme source. After 5 minutes of reaction at 37 ° C. using 5 mM 4-MU-α-galactopyranoside as a substrate, the reaction was stopped by adding 200 μl of a reaction stop solution (0.2 M glycine buffer (pH 10.7)). Measurement was performed with a fluorescent microplate reader (MTP-32 manufactured by Corona, Ex: 365 nm, Em: 450 nm). Enzyme activity was expressed as μmol of the substrate hydrolyzed in 1 hour per mg of protein (vertical axis in FIG. 6). The result is shown in FIG. FIG. 6 is a graph instead of a drawing showing the measurement results of α-galactosidase A activity. FIG. 6 shows that TIY20 strain has inactivated α-galactosidase, whereas C3-20, C3-7-2 and C4-1 show good α-galactosidase A activity, especially C3-20 and C3. It can be seen that -7-2 exhibits higher α-galactosidase A activity than wild-type W303-1A.

Mutant cdc6 — Production of Plasmid pREP1cdc6-1 Introducing DNA Fragment The mutant cdc6 (cdc6-1) DNA fragment was cloned into the BamHI-NotI site of the multicopy vector pREP1 for fission yeast forced expression as follows. Plasmid pREP1cdc6-1 (see FIG. 7 (a)) was constructed.
The amino acid sequence of fission yeast Cdc6 is shown in SEQ ID NO: 7, and the base sequence of DNA encoding Cdc6 is shown in SEQ ID NO: 8. The cdc6-1 mutant gene obtained in this example is obtained by replacing the 898th to 906th bases (GAT ATT GAA) in SEQ ID NO: 8 with GCCGGGCGCT. That is, a mutation was introduced such that the amino acid residue encoded by the nucleotide sequence from the 898th to the 906th base was replaced from DIE to AGA. The amino acid sequence of cdc6-1 thus obtained is shown in SEQ ID NO: 9, and the base sequence of the DNA encoding the cdc6-1 mutant gene is shown in SEQ ID NO: 10. Specifically, a mutant gene fragment of cdc6 was amplified by a site-directed method by PCR reaction using genomic DNA extracted from TN8 strain (h ⁹⁰ leu1-32) which is a wild strain of fission yeast as a template. For PCR reaction, forward primer (SEQ ID NO: 11): 5′-AGCTC GGATCC (BamHI) GATGACAGATAGGTCTCTCAAATGAGGCGCGTC-3 ′, reverse primer (SEQ ID NO: 12): 5′-TCGAGGCGACCTGCGCCAAGCGCCGGCAAAGCCTCATGAT-13 ′, primer 5′-AGCTCAGGATCATGAGCTTTGCCCGGCGCTTGCGCAGGTCGCA-3 ′, reverse primer (SEQ ID NO: 14): 5′-TCGAG GCGGCCGC (NotI) TCACCAGGACATTTCATCAAATCTTTTCA-3 ′ was used. By the reverse primer of SEQ ID NO: 12 and the forward primer of SEQ ID NO: 13, the above-described substitution of the 898th to 906th bases occurs. Both ends of the obtained amplified fragment were digested with restriction enzymes BamHI and NotI, and cloned into the BamHI-NotI site of the pREP1 vector (the plasmid thus obtained was named pREP1cdc6-1). This plasmid was able to express the mutant cdc6 gene (cdc6-1) under the control of the nmt1 promoter.

Obtaining a high-temperature-resistant strain of a sugar chain-modified strain Schizosaccharomyces pombe sugar chain-modified strain KT97 (h ^- leu1-32 ura4-D18 Δoch1 :: ura4 +) was transformed with the plasmid pREP1cdc6-1 obtained as described above. Transformed. KT97 (Yoko-o T et al., FEBS Letters 489, 75-80. (2001)) is disclosed in Japanese Patent Laid-Open No. 2001-161376. That is, KT97 is a gene disrupted strain of fission yeast in which the OCH1 gene is disrupted. The obtained transformant (KT97 <pREP1cdc6-1) was added to a fission yeast synthetic medium EMM (3 g physacid acid K +, 2.2 g NA ₂ HPO ₄ , 5 g NH ₄ Cl ₂ · 2H in order to introduce more mutations. ₂ O, 1 g KCl, 0.04 g Na ₂ SO ₄ , 1 mg pantothenic acid, 10 mg nicotinic acid, 10 mg myo-inositol, 1 mg biotin, 0.5 mg boric acid, 0.4 mg MnSO ₄ , 0.4 mg ZnSO ₄ · 7H _{2 O, 0.2mg FeCl 2 · 6H 2} O, 40mg molybdic acid, 0.1mg Kl, 40mg CuSO 4 · H 2 O, to 1mg Citric acid / 1L) (liquid), conditions can be as many divisions Control to the culture. In order to obtain a high-temperature resistant strain from these bacteria, it was cultured in an EMM solid medium at 37 ° C. for 6 days, and the colonies that had grown were caught.

Analysis of the sugar chain length added to the protein invertase in yeast To examine the N-linked sugar chain length of the four strains (C2-1, C2-2, C2-3, and C2-11) obtained in Example 6 The N-linked sugar chain length added to the acidic phosphatase produced in yeast was examined by the following method. Each strain was cultured in 5 ml of YEA medium (30 g glucose, 5 g Yeast extract, 20 g agarose, 0.16 g adenine-SO ₄ , 0.05 g Uracil), and then 5 ml of low phosphate YPD medium (10 g Yeast extract, 20 g Bacto Peptone, 10 mM MgSO ₄ , 20 g glucose / 1 L) was cultured overnight, and the bacteria were collected. Add the lysis buffer (62.5 mM Tris-HCl (pH 6.8), 1 mM EDTA, 10% glycerol, 0.1 mM DTT, 2 mM PMSF) and glass beads so that the collected bacteria become 20 OD / 50 μl, and cool. While crushed with a vortex mixer. The bacterial solution was centrifuged at 15,000 rpm for 10 minutes, and then the supernatant was collected. 7 μl of sample buffer (62.5 mM Tris-HCl (pH 6.8), 0.01% bromphenol blue, 15% glycerol) was added to 25 μl of the supernatant, and 10 μl of each was added to a 4-20% gradient gel. Electrophoresis (150 V, 2 hours) (upper layer buffer: 5.16 g Tris, 3.48 g glycine / 1 L; lower layer buffer: 14.5 g Tris, 0.024 N HCl / 1 L). The gel was shaken in 100 mM sodium acetate (pH 4.0) for 15 minutes, and the gel was pre-warmed to 37 ° C. (100 mM Na-acetate (pH 4.0), 24.6 mg 1-sodium naphthyl phosphate) Hydrate, 16.7 mg o-dianisidine, Tetrazotized / 50 ml) and incubated at 37 ° C. until color developed. The result is shown in FIG. FIG. 8 is an SDS gel electrophoresis photograph replacing the drawing for examining the N-linked sugar chain length. The lane in FIG. 8 shows JY741 (wild type), KT97 (parent strain), and C2-11 from the left. From FIG. 8, the N-linked sugar chain added to the acid phosphatase produced by the C2-11 strain obtained in Example 6 is compared with the wild strain (JY741), similar to the parent strain KT97. It was found to have a short single sugar chain length. The C2-11 strain was deposited with the patent microorganism. That is, if the C2-11 strain is used, a protein having a desirable sugar chain can be obtained. That is, the present invention also provides a protein production method using the C2-11 strain.

Analysis of Growth Recovery Efficiency Next, the growth recovery efficiency of the strain obtained in Example 6 was analyzed. After culturing with 5 ml of YEA at 30 ° C., it was transferred to 10 ml of YEA so that the OD600 was 0.1, and cultured at 30 ° C. Bacteria were collected at each time point and examined for OD600. The result is shown in FIG. FIG. 9 is a graph replaced with a drawing showing the recovery efficiency of growth at 30 ° C. FIG. 9 shows that the growth rate is higher than that of KT97 in all the analyzed strains.

  According to each of the above-mentioned Examples, novel budding yeast and fission yeast mutant yeast strains excellent in high-temperature resistance, growth ability and glycoprotein production ability can be obtained. As described above, since the mutant yeast strain effective in budding yeast and fission yeast can be obtained by using the method of the present invention, the present invention is limited to the budding yeast and fission yeast used in the above examples. In any case, a novel mutant yeast strain excellent in high temperature resistance, growth ability and glycoprotein production ability can be obtained.

Monosaccharide analysis of yeast cell wall Yeast C4-1 and C3-7-2 obtained in Example 1 were permeabilized with 5 ml of YPD at 30 ° C. for 15 hours, and then centrifuged (4000 rpm for 5 minutes). The cells were collected by suspending in 1 ml of 10 mM Tris-HCl buffer (pH 7.5, 1 mM PMSF) and centrifuging (4000 rpm for 5 minutes). The cells were washed by performing this operation three times. The suspension was resuspended in 0.1 ml of the above Tris-HCl buffer, glass beads were added to the liquid surface, and the mixture was stored at -20 ° C for 1 hour. After disrupting the cells with a vortex mixer, only the cell disruption solution was recovered, and the pellet was recovered by centrifugation (1000 g, 10 minutes). The pellet (cell wall) was suspended in 1 ml of 1M NaCl and centrifuged. By repeating this operation three times, the pellet was washed. Next, it was washed 3 times with 1 ml of 1 mMPMSF. Resuspended in 0.2 ml 1mMPMSF.
50 μl of sterile ultrapure water was added to 50 μl of the cell wall suspension, 100 μl of 4M TFA was added, and the mixture was incubated at 100 ° C. for 4 hours. After completely evaporating the solvent in a vacuum dryer, 100 μl of 0.2 M ammonium acetate and 10 μl of acetic acid were added and incubated at room temperature for 30 minutes. After drying in a vacuum dryer, 100 μl of 0.2M ammonium acetate and 10 μl of acetic acid were added and incubated at room temperature for 30 minutes. The suspension was transferred to a PA tube and dried. The PA labeling was performed using a PA labeling kit (PALSTATION Pyridylation Reagent Kit for monosaccharide analysis (TaKaRa)). HPLC was carried out at a column: TSK-GEL SUGAR AX type (TOSHO), solvent: 0.7 M potassium borate (pH 9.0); acetonitrile = 9: 1, 65 ° C., flow rate: 0.3 ml / min. The composition ratio of monosaccharides was calculated from the peak area ratio (FIG. 10). FIG. 10 is a graph replaced with a drawing showing the analysis result of the monosaccharide content in the yeast cell wall. The vertical axis represents%, GlcNAc represents N-acetylglucosamine, Glc represents glucose, and Man represents mannose.
As shown in FIG. 10, the proportion of glucose in the TIY20 strain was as high as about 80% compared to the proportion of glucose in the WT (about 45%). The strain -7-2 showed a higher glucose ratio (90% or more). Although it has been considered technically very difficult to produce a yeast having a high β-glucan content such that the proportion of glucose exceeds about 80%, the yeast of the present invention further has about 10 % Or more of the glucose was improved. From the above, it can be said that the yeast of the present invention is extremely useful in terms of productivity, cost, etc., in β-glucan production technology using yeast.

  The yeast avoiding high-temperature sensitivity or recovering its growth ability according to the present invention and a method for producing the same can obtain a yeast excellent in high-temperature resistance, growth ability and glycoprotein production ability, not only budding yeast and fission yeast, but also yeast Can be widely applied in general. The yeast that produces β-glucan of the present invention with high efficiency and a method for producing the same can obtain a yeast that is more excellent in β-glucan production ability than conventionally known yeasts. It can be widely applied to yeast in general. The present invention can be used in the pharmaceutical industry because it can be effectively used for production of glycoprotein and β-glucan using yeast. Moreover, since identification of gene disruption strains and the like can be controlled as appropriate, it can also be used in the reagent industry.

FIG. 1 is a diagram for studying recovery of high-temperature resistance of the plasmid pAB100 used in Example 1 and strains into which the plasmid was introduced. FIG. 1 (a) is a diagram showing an outline of plasmid pAB100, FIG. 1 (b) is a photograph replacing a drawing showing the recovery of high-temperature resistance of the strain, and FIG. It is a figure which shows the fraction of b). FIG. 2 is an SDS gel electrophoresis photograph replacing the drawing for examining the N-linked sugar chain length. FIG. 3 is a graph instead of a drawing showing the growth recovery efficiency at 30 ° C. and 37 ° C. FIG. 4 is a graph showing the results of column analysis instead of a drawing for analyzing a sugar chain structure. FIG. 5 is a graph replaced with a drawing for showing the secretion efficiency of the protein secreted from the mutant strain. FIG. 6 is a graph instead of a drawing showing the measurement results of α-galactosidase A activity. FIG. 7 is a diagram for examining the recovery of the viability of the plasmid pREP1cdc6-1 used in Example 6 and the strain introduced with the plasmid. FIG. 7 (a) is a diagram showing an outline of plasmid pREPcdc6-1, FIG. 7 (b) is a photograph replacing a drawing showing recovery of high-temperature resistance of the strain, and FIG. 7 (c) is a diagram showing FIG. It is a figure which shows the fraction of (b). FIG. 8 is an SDS gel electrophoresis photograph replacing the drawing for examining the N-linked sugar chain length in Example 7. FIG. 9 is a graph replaced with a drawing showing the recovery efficiency of growth at 30 ° C. in Example 7. FIG. 10 is a graph replaced with a drawing showing the analysis result of the monosaccharide content in the yeast cell wall. The vertical axis represents%, GlcNAc represents N-acetylglucosamine, Glc represents glucose, and Man represents mannose.

Sequence number 5: Primer sequence number 6: Primer sequence number 11: Primer sequence number 12: Primer sequence number 13: Primer sequence number 14: Primer

Claims (8)

A yeast obtained by a method for producing a yeast that avoids high-temperature sensitivity or restores growth, comprising a step of controlling a proofreading function of a DNA polymerase in a yeast gene function-deficient strain,
The yeast gene function-deficient strain is a gene-disrupted strain having the ability to produce glycoprotein introduced with och1 disruption,
The yeast is a fission yeast having a mutant DNA polymerase in which the GAT ATT GAA at the 898th to 906th bases in the cdc6 + gene represented by SEQ ID NO: 8 is substituted with GCC GGC GCT.   Yeast deposited under the accession number “FERM BP-11124” at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Controlling the proofreading function of DNA polymerase in a yeast gene function deficient strain,
The yeast gene function-deficient strain is a yeast mutant strain capable of producing a glycoprotein having a mammalian sugar chain,
The step of controlling the proofreading function of the DNA polymerase is a method in which the 498th to 906th bases GAT ATT GAA in the cdc6 + gene represented by SEQ ID NO: 8 are replaced with GCC GGC GCT in the yeast gene function deficient strain. Including the step of transforming
A yeast obtained by a method for producing a yeast having the ability to express foreign genes,
The yeast gene function-deficient strain is a gene-disrupted strain having the ability to produce glycoprotein introduced with och1 disruption,
The yeast is a fission yeast having a mutant DNA polymerase in which the GAT ATT GAA at the 898th to 906th bases in the cdc6 + gene represented by SEQ ID NO: 8 is substituted with GCC GGC GCT.   A glycoprotein which is obtained by culturing the fission yeast according to any one of claims 1 to 3 in a medium, generating a glycoprotein having a mammalian sugar chain, and collecting the glycoprotein having a mammalian sugar chain from the culture. Production method.   A method for producing β-glucan, wherein the fission yeast according to any one of claims 1 to 3 is cultured in a medium to produce β-glucan, and the β-glucan is collected from the culture. A method for producing fission yeast that avoids high-temperature sensitivity or restores growth, comprising a step of controlling a DNA polymerase proofreading function in a gene function deficient strain of fission yeast,
The fission yeast gene function-deficient strain is a gene-disrupted strain having glycoprotein production ability into which och1 disruption is introduced,
The fission yeast has a mutant DNA polymerase in which the GAT ATT GAA at the 898th to 906th bases in the cdc6 + gene represented by SEQ ID NO: 8 is replaced with GCC GGC GCT.
A method for producing fission yeast.   A fission yeast obtained by the method for producing fission yeast according to claim 6 is cultured in a medium to produce a glycoprotein having a mammalian sugar chain, and the glycoprotein having a mammalian sugar chain is collected from the culture. A method for producing glycoprotein.   A method for producing β-glucan, comprising culturing fission yeast obtained by the method for producing fission yeast according to claim 6 in a medium, generating β-glucan, and collecting the β-glucan from the culture.

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