# Patent

## Title
Transformant of Schizosaccharomyces pombe mutant and cloning vector

## Abstract

Provided is a transformant of S. pombe mutant which can produce and collect β-glucosidase without requiring complicated separation steps, and a vector which is useful for transforming a yeast of the genus Schizosaccharomyces. The transformant of a S. pombe mutant of the present invention is a transformant of a Schizosaccharomyces pombe mutant which exhibits an increased Gsf activity and decreased or no pyruvyl transferase Pvg1 enzymatic activity, and is comprised of a structural gene sequence encoding a β-glucosidase derived from a filamentous fungus, and a promoter sequence and a terminator sequence for expressing the structural gene in a chromosome or as an extrachromosomal gene. Further, the present invention relates to a cloning vector which is characterized by comprising a hsp9 gene promoter or an ihc1 gene promoter of a yeast of the genus Schizosaccharomyces, and is useful for transforming a yeast of the genus Schizosaccharomyces, an expression vector, a transformant, etc.

## Background
TECHNICAL FIELD
The present invention relates to a transformant of a Schizosaccharomyces pombe mutant which exhibits non-sexual flocculation and can produce β-glucosidase, and a vector useful for transforming a yeast of the genus Schizosaccharomyces. 
BACKGROUND ART
To produce biomass fuels including a sugar as a fermentation feedstock, a bioethanol, etc. from a cellulosic biomass such as a wood, rice straw, rice husk or weed, it is required to degrade the main structural component of a plant cell wall, cellulose. To degrade cellulose, an acid saccharification method such as a concentrated sulfuric acid saccharification method or a dilute sulfuric acid saccharification method, an enzymatic saccharification method, etc. may be employed. Because of recent development in biotechnology, research and development of an enzymatic saccharification method are actively carried out.
In the enzymatic saccharification of cellulose, enzymes collectively known as cellulases are utilized. Firstly, an endoglucanase (EG), which has an activity to cleave cellulose chains at random, degrades an amorphous region of cellulose to expose terminal glucose residues. The exposed glucose residues are degraded by a cellobiohydrolase (CBH) to release cellobiose. Thereafter, the released cellobiose is degraded by β-glucosidase (BGL) to release glucose.
For the saccharification of cellulose, filamentous fungi of the genus Aspergillus and the genus Trichoderma are widely used, since they can produce various cellulases and hemicellulases which are required for degrading and saccharifying a crystalline cellulose, and they can secrete a large amount of such enzymes to their extracellular environment.
Further, it has been tried to express such cellulases of filamentous fungi in a heterologous microorganism. Non-patent document 1 discloses that a budding yeast Saccharomyces cerevisiae was transformed with a gene encoding β-glucosidase 1 (BGL 1) of Aspergillus aculeatus to obtain a transformant, and the obtained transformant expressed such an enzyme.
However, in the enzymatic saccharification method, as the enzymatic hydrolysis of cellulose proceeds, glucose accumulates in the reaction system and the accumulated glucose inhibits β-glucosidase, whereby accumulation of cellobiose proceeds. Further, there is a problem such that the complete degradation of cellulose may not be achieved since the accumulated cellobiose inhibits endoglucanase and cellobiohydrolase. Accordingly, development of a highly functional β-glucosidase has been desired.
On the other hand, the genetic analysis of a yeast of the genus Schizosaccharomyces is more advanced than that of a filamentous fungus of the genus Aspergillus or the genus Trichoderma, and the yeast has a lot of advantages like availability of various useful mutants and gene transfer vectors, and its suitability for industrial large-scale production of a protein. However, the yeast of the genus Schizosaccharomyces does not have endogenous β-glucosidase gene, whereby it cannot utilize cellobiose. The present inventors transformed a yeast of the genus Schizosaccharomyces with a gene encoding β-glucosidase thereby to express the enzyme from the obtained transformant (Patent Document 1).
Further, in Non-Patent Document 1, there is no description about an inhibitory effect of glucose to β-glucosidase produced by a budding yeast.
On the other hand, for recovering a β-glucosidase secreted from the transformant, a step of separating a culture broth and cells is required. Although steps of centrifugation, continuous centrifugation, membrane separation, etc. may be mentioned as the separation step, all of them are complicated steps and require a tremendous amount of labor and time.
In addition, it is easily expected that the complexity of the separation step increases as the scale of β-glucosidase production expands.
On the other hand, in the case of using a yeast which exhibits non-sexual flocculation (a property of aggregating non-sexually), aggregated yeast cells are easily separated from a culture broth after cultivation, whereby it is preferred to use a yeast which exhibits non-sexual flocculation as a host cell in the β-glucosidase production.
As the yeast which exhibits non-sexual flocculation, FLO mutants are known for budding yeast Saccharomyces cerevisiae. Further, mutants which exhibit non-sexual flocculation have been reported (e.g. Patent Document 2) for fission yeast Schizosaccharomyces pombe (hereinafter also referred to as S. pombe).
Further, a yeast of the genus Schizosaccharomyces such as S. pombe is phylogenetically quite different from budding yeast Saccharomyces cerevisiae. It is significantly different from other yeasts in its chromosome structure and various mechanisms including genome replication mechanism, RNA splicing mechanism, transcriptional machinery and post-translational modification, and some of them are known to be similar to those of animal cells. Therefore, it has been widely used as a model eukaryote (Non-Patent Document 2).
Because of its various characteristics, S. pombe is considered as a unicellular eukaryote closer to higher animal cells and is a very useful yeast as a host for expression of foreign genes, especially genes derived from higher animals. In particular, it is known to be suitable for expression of genes derived from animals such as human (Patent Documents 3 to 9).
For expressing a protein derived from a foreign structural gene by using S. pombe hosts, usually, a promoter which promotes transcription of the foreign structural gene encoding the protein is required. As the promoter, endogenous promoters for S. pombe genes and promoters from other organisms or viruses are known.
As promoters which have been utilized for the protein expression in S. pombe hosts, promoters endogenous to S. pombe including an alcohol dehydrogenase (adh1) gene promoter, a nmt1 gene promoter involved in thiamine metabolism, a fructose-1, 6-bis phosphatase (fbp1) gene promoter involved in glucose metabolism, an invertase (inv1) gene promoter involved in catabolite repression (Patent Document 7 or 10), a heat shock protein gene promoter (Patent Document 11) may, for example, be mentioned. Further, a promoter of a virus such as hCMV, SV40, or CaMV (constructive expression) is also known (Patent Document 4, 6 or 12).
PRIOR ART DOCUMENTS
Patent Documents


    Patent Document 1: WO 2012/060389
    Patent Document 2: JP-A-2000-106867
    Patent Document 3: JP-B-2776085
    Patent Document 4: JP-A-H07-163373
    Patent Document 5: WO 96/23890
    Patent Document 6: JP-A-H10-234375
    Patent Document 7: JP-A-H11-192094
    Patent Document 8: JP-A-2000-136199
    Patent Document 9: JP-A-2000-262284
    Patent Document 10: WO 99/23223
    Patent Document 11: WO 2007/26617
    Patent Document 12: JP-A-H5-15380


Non-Patent Documents


    Non-Patent Document 1: G. Takada, et al., Biosci. Biotechnol. Biochem., 62(8), 1615-1618, 1998.
    Non-Patent Document 2: Giga-Hama and Kumagai, eds., Foreign gene expression in fission yeast Schizosaccharomyces pombe, Springer-Verlag, (1997).


DISCLOSURE OF INVENTION
Technical Problem
During the cultivation of yeasts, the pH of a culture broth may be shifted to an acidic range. Particularly, in the case of expressing a large amount of acidic secretory proteins, the pH of a culture broth is likely to be shifted to an acidic range of from 2 to 5 at the end of cultivation. Further, when cultivating yeasts, the optimum pH for the cultivation may be lower than 5, considering productivity of a desired protein.
Therefore, in the case of using yeasts which exhibit flocculation in a culture broth having a relatively high pH, e.g. higher than pH 5, and do not aggregate under an acidic condition of pH 2 to 5, for the aggregation of the yeasts, a neutralization process is required to be carried out at the end of cultivation so as to adjust the pH to a neutral range.
Yeasts disclosed in Patent Document 1 are S. pombe mutants which exhibit non-sexual flocculation, and are preferred as host cells for the above-described expression system. However, although such S. pombe mutants were found to aggregate non-sexually in a YPD medium (usually, pH 5.6 to 6.0), it is unclear as to whether they exhibit sufficient flocculation under an acidic condition.
Therefore, the present invention is to provide a transformant of a S. pombe mutant which can produce and collect β-glucosidase without requiring complicated separation steps, and a method for producing β-glucosidase by using the transformant.
Further, the present invention is to provide an expression vector which is associated with a novel promoter and can express a protein derived from a foreign structural gene efficiently by genetic engineering when a yeast of the genus Schizosaccharomyces is used as a host; a cloning vector for preparing the expression vector; a method for producing the expression vector; a transformant comprising the expression vector; a method for producing the transformant; and a method for producing a protein using the transformant.
Solution to Problem
The transformant of a S. pombe mutant of the present invention is a transformant of a Schizosaccharomyces pombe mutant which exhibits an increased Gsf activity and decreased or no pyruvyl transferase Pvg1 enzymatic activity, and is comprised of a structural gene sequence encoding a β-glucosidase derived from a filamentous fungus, and a promoter sequence and a terminator sequence for expressing the structural gene in a chromosome or as an extrachromosomal gene.
Further, in the transformant of a S. pombe mutant of the present invention, the β-glucosidase is preferably BGL1.
Further, in the transformant of a S. pombe mutant of the present invention, the filamentous fungus is preferably a microorganism of the genus Aspergillus. 
Further, in the transformant of a S. pombe mutant of the present invention, the β-glucosidase is preferably comprised of an amino acid sequence represented by SEQ ID NO: 1 or is comprised of the amino acid sequence having deletion, substitution or addition of at least one amino acid, and has a catalytic activity to hydrolyze a β-D-glucopyranoside bond.
Further, the method for producing a β-glucosidase of the present invention is characterized by cultivating the above-mentioned transformant and, from a cell or a culture supernatant thereby obtained, recovering a β-glucosidase.
Hereinafter, the invention relates to a transformant comprising a structural gene sequence encoding the β-glucosidase is referred to as the invention of the first embodiment.
The cloning vector of the present invention is characterized by comprising a hsp9 gene promoter or an ihc1 gene promoter of a yeast of the genus Schizosaccharomyces, a cloning site for introducing a foreign structural gene which is located downstream from the promoter and is governed by the promoter, and a terminator capable of functioning in the yeast of the genus Schizosaccharomyces. 
The promoter of hsp9 gene (hereinafter also referred to as hsp9 promoter) is preferably a region containing 1 to 400 bp upstream from the 5′ end of a hsp9 gene ORF (open reading frame), and is more preferably one comprised of a nucleotide sequence represented by SEQ ID NO: 6 or the nucleotide sequence having deletion, substitution or addition of at least one nucleotide, and has a promoter activity.
The promoter of ihc1 gene (herein after also referred to as ihc1 promoter) is preferably a region containing 1 to 501 bp upstream from the 5′ end an ihc1 gene ORF (open reading frame), and is more preferably one comprised of a nucleotide sequence represented by SEQ ID NO: 9 or the nucleotide sequence having deletion, substitution or addition of at least one nucleotide, and has a promoter activity.
The method for producing an expression vector of the present invention is characterized by introducing a foreign structural gene into a cloning site of the above-described cloning vector, and the expression vector of the present invention is an expression vector comprising a foreign structural gene which is introduced into the cloning site of the above-described cloning vector.
The transformation method for a yeast of the genus Schizosaccharomyces of the present invention is characterized by introducing the above-described expression vector into the yeast of the genus Schizosaccharomyces, and the transformant of the present invention is a transformant comprising the above-described expression vector.
The method for producing a protein of the present invention is characterized by cultivating the above-described transformant and, from a cell or a culture supernatant thereby obtained, recovering a protein encoded by the above-described foreign structural gene.
Hereinafter, the invention relates to a cloning vector, an expression vector and a transformant having the above-described hsp9 promoter or ihc1 promoter, and the invention relates to a method for producing the expression vector, a method for producing a transformant and a method for producing a protein are referred to as the inventions of the second embodiment.
Advantageous Effect of Invention
According to the transformant of a S. pombe mutant of the present invention, it is possible to produce and collect a β-glucosidase without requiring complicated separation steps.



BRIEF DESCRIPTION OF DRAWING(S)
FIG. 1 is a construction map of expression vector pSL6AaBGL1.
FIG. 2 is a construction map of expression vector pSL6P3AaBGL1.
FIG. 3 is a construction map of expression vector pSL14P3AaBGL1.
FIG. 4 is a construction map of expression vector pUC19-ura4.
FIG. 5 is a graph showing the cell growth (OD660 values) of a normal strain or a flocculation strain in Test Example 8.
FIG. 6 (a) is a graph showing the glucose concentration of a culture broth for a normal strain or a flocculation strain in Test Example 8. FIG. 6 (b) is a graph showing the ethanol concentration of a culture broth for a normal strain or a flocculation strain in Test Example 8.
FIG. 7 is a graph showing the pNPG degradation activity of an AaBGL1 of a normal strain or a flocculation strain in Test Example 9.
FIG. 8 is a graph showing the cell growth (OD660 values) of a normal strain or a flocculation strain in Test Example 10.
FIG. 9 (a) is a graph showing the glucose concentration of a culture broth for a normal strain or a flocculation strain in Test Example 10. FIG. 9 (b) is a graph showing the ethanol concentration of a culture broth for a normal strain or a flocculation strain in Test Example 10.
FIG. 10 is a photograph illustrating a sedimentation status of a normal strain or a flocculation strain in Test Example 11.
FIG. 11 is a photograph illustrating microscopic observation results of a normal strain or a flocculation strain in Test Example 11.
FIG. 12 is a graph showing the cell growth (OD660 values) at 30° C. or 34° C. in Test Example 12.
FIG. 13A is a graph showing the glucose concentration of a culture broth at 30° C. or 34° C. in Test Example 12.
FIG. 13B is a graph showing the ethanol concentration of a culture broth at 30° C. or 34° C. in Test Example 12.
FIG. 14 is a graph showing the pNPG degradation activity of an AaBGL1 at 30° C. or 34° C. in Test Example 12.
FIG. 15 is a graph showing the results of SDS-PAGE of Test Example 13.
FIG. 16 is a construction map of expression vector pSL14-EGFP.
FIG. 17 is a construction map of expression vector pSL6-EGFP.
FIG. 18 is a graph showing the results of measuring the fluorescence intensity (relative value obtained by taking the fluorescence intensity of a SL6E strain culture broth as 1) of each culture broth in Test Example 14.
FIG. 19 is a construction map of multiple cloning vector pSL6.
FIG. 20 is a construction map of multiple cloning vector pSL12.
FIG. 21 is a construction map of multiple cloning vector pSL17.
FIG. 22 is a construction map of expression vector pSL12-EGFP.
FIG. 23 is a graph showing the results of measuring the time-course changes in [fluorescence intensity/OD600] of each culture broth in Test Example 15.
FIG. 24 is a construction map of multiple cloning vector pSL9.
FIG. 25 is a construction map of multiple cloning vector pSL14.
FIG. 26 is a construction map of multiple cloning vector pSL14lacZ.



DESCRIPTION OF EMBODIMENTS
In the present specification, “foreign structural gene” is a gene contained in an expression vector and encodes a protein, and may be a structural gene endogenous to a host to be introduced with the expression vector or a structural gene of an organism heterologous to the host.
In the present specification, “protein derived from a foreign structural gene” is a protein derived from a foreign structural gene and produced from a transformant, and hereinafter, it is also referred to as “foreign protein”. Further, it is also referred to as a heterologous protein in a case where the foreign structural gene is a structural gene of an organism heterologous to the host.
Firstly, the first embodiment of the present invention will be described.
[S. pombe Mutant]
In the present invention, a S. pombe mutant to be used as a host of a S. pombe mutant transformant is a mutant exhibits, as a result of modifications in at least a part of S. pombe genes, an increased Gsf activity and decreased or no pyruvyl transferase Pvg1 enzymatic activity. In S. pombe, when the Gsf activity increases and the enzymatic activity of Pvg1 decreases or is lost, a property aggregating non-sexually even under an acidic condition will be obtained. Hereinafter, a property of aggregating non-sexually under an acidic condition is also referred to as “acid-resistance non-sexual flocculation”.
In other words, the S. pombe mutant of the present invention is a mutant which has acquired acid-resistance non-sexual flocculation.
In the present invention, “non-sexual flocculation” is an aggregating property different from the property of aggregating sexually which is intrinsic to S. pombe (sexual flocculation). However, it does not mean that the intrinsic sexual flocculation is lost. Further, “constitutive flocculation” is a property identical to the non-sexual flocculation, and particularly refers to a property of aggregating (non-sexually) concurrently with the cell growth at a growing stage.
In the present invention, “Gsf activity” means non-sexual flocculation exhibited in the pH (e.g. pH 5 to 6) generally used for cultivating S. pombe. Further, a gene related to the Gsf activity is referred to as a flocculation gene.
gsf2 gene is a flocculation gene in S. pombe. The systematic name of gsf2 gene of S. pombe is SPCC1742.01.
Pvg1 is a pyruvate transferase. The systematic name of pvg1 gene encoding Pvg1 of S. pombe is SPAC8F11.10c.
Further, the entire nucleotide sequence of the chromosomes of S. pombe is stored and opened to the public in “Schizosaccharomyces pombe Gene DB (HyperText Markup Language://WorldWideWeb.genedb.org/genedb/pombe/)” of Sanger Institute. The sequence data of S. pombe genes described in the present specification are available from the data base by searching with a gene name or a systematic name.
S. pombe originally exhibits sexual flocculation induced by pheromones. For example, during the growth process, nutritional deficiencies likely to cause sexual flocculation. However, in an artificial large-scale cultivation such as a tank culture, sexual flocculation is usually less likely to occur since cultivation is carried out with a culture broth containing a sufficient amount of nutrients. On the other hand, since the S. pombe mutant used in the present invention exhibits non-sexual flocculation, it is subject to flocculation (constitutive flocculation) even in the case of cultivating in a culture broth containing a sufficient amount of nutrients.
The Gsf activity of a S. pombe mutant can be increased by, for example, increasing the expression amount of gsf2 gene. Further, the enzymatic activity of Pvg1 of S. pombe can be decreased or inactivated by deleting pvg1 gene encoding Pvg1 or by introducing a mutation which causes decrease or inactivation of the enzymatic activity of Pvg1 into the gene.
Therefore, the S. pombe mutant used in the present invention can be produced by employing a S. pombe which is prepared via a genetic engineering method and does not exhibit acid-resistance non-sexual flocculation of wild-type strains, etc. as a host, integrating a foreign gsf2 gene, and deleting a gene encoding Pvg1 or introducing a mutation which causes decrease or inactivation of the enzymatic activity of Pvg1 into the gene.
The expression amount of gsf2 gene can be increased by integrating a foreign gsf2 gene via a genetic engineering method. Introduction of a new gsf2 gene into a host is important, and a gsf2 gene to be introduced may be homologous to an endogenous gsf2 gene of the host, or may be a gsf2 gene derived from a heterologous organism.
As a method for introducing gsf2 gene into a host via a genetic engineering method, publicly known methods can be used. As a method for introducing a foreign structural gene into a S. pombe host, methods disclosed in JP-A-5-15380, WO 95/09914, JP-A-10-234375, JP-A-2000-262284, JP-A-2005-198612, WO 2010/087344, etc. may be used.
It is preferred to introduce gsf2 gene into the chromosome of S. pombe. By introducing gsf2 gene into a chromosome, a transformant having a high passage stability can be obtained. Further, multiple copies of gsf2 gene can be introduced into a chromosome. By introducing multiple copies of gsf2 gene, the expression efficiency of gsf2 gene can be increased. In a S. pombe mutant, the copy number of gsf2 gene integrated into the chromosome is preferably from 1 to 20, more preferably from 1 to 8.
As a method for introducing gsf2 gene into a chromosome, publicly known methods can be used. For example, by the method disclosed in JP-A-2000-262284, multiple copies of gsf2 gene can be introduced into a chromosome. Further, single copy of gsf2 gene can be introduced into a chromosome. Further, as described below, single or multiple copies of gsf2 gene can be introduced into multiple sites on a chromosome.
The method for introducing gsf2 gene into the chromosome of S. pombe is preferably a homologous recombination method using a vector comprising an expression cassette containing gsf2 gene and a recombination region (hereinafter referred to as gsf2 vector).
The gsf2 vector comprises an expression cassette containing gsf2 gene and a recombination region.
The expression cassette is a combination of DNA necessary for expressing Gsf2, and contains gsf2 gene, a promoter which functions in S. pombe and a terminator which functions in S. pombe. Further, it may contain at least one of a 5′-untranslation region and a 3′-untranslation region. Further, it may contain an auxotrophic complementation marker. The expression cassette is preferably an expression cassette containing gsf2 gene, a promoter, a terminator, a 5′-untranslation region, a 3′-untranslation region, and an auxotrophic complementation marker. Multiple copies of gsf2 gene may be present in the expression cassette. The copy number of gsf2 gene in the expression cassette is preferably from 1 to 8, more preferably from 1 to 5.
The promoter and terminator which function in S. pombe may be those which can maintain expression of Gsf2 by functioning in the transformant even under an acidic condition. As the promoter which functions in S. pombe, a promoter endogenous to S. pombe (preferably one having a high transcriptional activity), or a promoter exogenous to S. pombe (such as a promoter derived from a virus) may be used. Further, two or more types of promoters may be contained in the vector.
As the promoter endogenous to S. pombe, an alcohol dehydrogenase gene promoter, a nmt1 gene promoter involved in thiamine metabolism, a fructose 1,6-bisphosphatase gene promoter involved in glucose metabolism, an invertase gene promoter involved in catabolite repression (WO99/23223) or a heat shock protein gene promoter (WO2007/26617) may, for example, be mentioned. As the promoter exogenous to S. pombe, promoters derived from an animal cell virus disclosed in JP-A-5-15380, JP-A-7-163373 and JP-A-10-234375 may, for example, be mentioned. Among these promoters, a nmt1 gene promoter or its modified promoter (nmt1+, nmt41 or the like) having a high expression efficiency, a hCMV promoter and a SV40 promoter are preferred.
Further, the below-described hsp9 promoter or ihc1 promoter for the second embodiment of the present invention may also be used.
As the terminator which functions in S. pombe, a terminator endogenous to S. pombe or a terminator exogenous to S. pombe may be used. Further, two or more types of terminators may be contained in the vector.
As the terminator, terminators derived from human disclosed in JP-A-5-15380, JP-A-7-163373 and JP-A-10-234375 may, for example, be mentioned, and human lipocortin-1 terminator is preferred.
The recombination region of the vector is a region having a nucleotide sequence which can induce homologous recombination with a target site in the chromosome of S. pombe at which homologous recombination is to be achieved. Further, the target site is a site to become a target for integration of an expression cassette in the chromosome of S. pombe. The target site can be designed freely by letting the recombination region of the vector have a nucleotide sequence which induces homologous recombination with the target site.
The recombination region is required to have a nucleotide sequence homology of at least 70% with the nucleotide sequence of the target site. Further, the nucleotide sequence homology between the recombination region and the target site is preferably at least 90%, more preferably at least 95%, in view of increasing the efficiency of homologous recombination. By using a vector having such a recombination region, the expression cassette is integrated into the target site by homologous recombination.
The length (number of base pairs) of the recombination region is preferably from 20 to 2,000 bp. When the length of the recombination region is at least 20 bp, homologous recombination is likely to be induced. Further, when the length of the recombination region is at most 2,000 bp, reduction in the homologous recombination efficiency due to too large vector size is likely to be prevented. The length of the recombination region is preferably at least 100 bp, more preferably at least 200 bp. Further, the length of the recombination region is preferably at most 800 bp, more preferably at most 400 bp.
The vector may contain another DNA region in addition to the above-described expression cassette and recombination region. For example, a replication origin region called “ori” which is necessary for replication in E. coli, an antibiotic resistance gene (neomycin resistance gene or the like), etc. may be mentioned. These are genes generally required for the construction of a vector using E. coli. The replication origin region is preferably removed when integrating the vector into the chromosome of the host, as described below.
The vector is a vector having a circular DNA structure or a linear DNA structure, and is preferably introduced into S. pombe cells in the form of a linear DNA structure. That is, in a case where a vector having a circular DNA structure like a usual plasmid DNA is used, the vector is preferably cut open to a linear form by a restriction enzyme before its introduction into the S. pombe cells.
In this case, the vector having a circular DNA structure is cut open at a position within the recombination region. The resulting vector has parts of the recombination regions exist at both ends and is integrated entirely into the target site of a chromosome by homologous recombination.
The vector may be constructed by other methods without cutting a vector having a circular DNA structure so long as a linear DNA structure having parts of the recombination region at both ends can be obtained.
As the vector, a plasmid derived from E. coli such as pBR322, pBR325, pUC118, pUC119, pUC18, pUC19 or the like may suitably be used.
In this case, it is preferred that the replication origin region called “ori” required for replication in E. coli is removed from the plasmid vector to be used for homologous recombination. Thus, the integration efficiency at the time of integrating the above-described vector into a chromosome can be increased.
The method for constructing the vector in which the replication origin region is removed is not particularly limited, but is preferably the method disclosed in JP-A-2000-262284. That is, it is preferable to preliminarily construct a precursor vector carrying the replication origin region at a position to be cut within the recombination region so that the replication origin region will be cut-off from the vector at the time of preparing a linear DNA structure. Thus, a vector in which the replication origin region is removed can be obtained easily.
Further, it may be a method wherein a precursor vector containing an expression cassette and a recombination region is constructed by using the expression vectors and their construction methods disclosed in JP-A-5-15380, JP-A-7-163373, WO96/23890, JP-A-10-234375, and then the replication origin region is removed from the precursor vector by using a usual genetic engineering method to obtain a vector to be used for homologous recombination.
The target site for integration of the vector may be present in only one position of the chromosome of S. pombe, or may be present in two or more positions thereof. When the target site is present in two or more positions, the vector can be integrated into two or more positions of the chromosome of S. pombe. Further, when the vector has multiple copies of gsf2 gene, multiple copies of gsf2 gene can be integrated into one position of the target site. Further, the expression cassette may be integrated into two or more types of target sites by using two or more types of vectors having recombination regions corresponding to the respective target sites.
When the expression cassette is integrated into one target site, the target site disclosed in JP-A-2000-262284 may, for example, be used. By using two or more types of vectors having different recombination regions, each of the vectors can be integrated into different target sites. However, this method becomes complicated in the case of integrating vectors into two or more positions of the chromosome.
Assuming that nucleotide sequences which are substantially identical to one another and present in plural positions of a chromosome can be used as target sites and vectors can be integrated into the respective plural positions of target sites, vectors can be integrated into two or more positions of the chromosome by using a single type of vector. The nucleotide sequences which are substantially identical to one another means that the homology between the nucleotide sequences is at least 90%. The homology among the target sites is preferably at least 95%. Further, the length of the nucleotide sequences which are substantially identical to one another is a length encompassing the recombination region of a vector, and is preferably at least 1,000 bp. As compared with a case wherein multiple copies of gsf2 gene are integrated into one target site, even if the integration numbers of gsf2 gene are the same, when gsf2 gene is integrated into plural target sites in a dispersed manner, drop-out of every gsf2 gene from the chromosome is less likely to occur during cultivation, whereby the maintenance stability during cultivation of the transformant increases.
The target site present in plural positions of the chromosome is preferably transposon gene Tf2. Tf2 is a transposon gene which exists in every three chromosomes (monoploid) of S. pombe at 13 positions in total and has a length (number of base pairs) of about 4,900 bp, with a nucleotide sequence homology of 99.7% (refer to the below-identified reference).
Nathan J. Bowen et al., “Retrotransposons and Their Recognition of pol II Promoters: A Comprehensive Survey of the Transposable Elements From the Complete Genome Sequence of Schizosaccharomyces pombe”, Genome Res., 2003 13: 1984-1997.
It is possible to integrate a vector into only one position of Tf2 which exists in 13 positions of the chromosome. In such a case, by integrating a vector containing two or more copies of gsf2 gene, a transformant having two or more copies of gsf2 gene can be obtained. Further, by integrating a vector into two or more positions of Tf2, a transformant having two or more copies of gsf2 gene can be obtained. In this case, by integrating a vector containing two or more copies of gsf2 gene, a transformant having even more copies of gsf2 gene can be obtained. When a vector is integrated into all 13 positions of Tf2, there is a possibility that the load on the survival and growth of the transformant becomes too large. It is preferred that a vector is integrated into at most 8 positions out of the 13 Tf2 positions, and it is more preferred that a vector is integrated into at most 5 positions.
For producing the S. pombe mutant of the present invention by a genetic engineering method, S. pombe having a marker for selecting a transformant is preferably used as a host. For example, it is preferred to use a host which essentially requires a specific nutrient factor for its growth due to deletion of a certain gene. By using a vector carrying the deleted gene (auxotrophic complementation marker), a transformant lacking the auxotrophy of the host will be obtained.
It is possible to select the transformant by using the difference in auxotrophy between the host and the transformant.
For example, a S. pombe host which has been made auxotrophic for uracil by deletion or inactivation of orotidine phosphate decarboxylase (ura4 gene) is transformed with a vector containing ura4 gene (auxotrophic complementation marker), and transformants carrying the vector are obtained by selecting ones lacking uracil auxotrophy. The gene to be deleted to make an auxotrophic host is not limited to ura4 gene when it is used for selection of a transformant, and may, for example, be isopropyl malate dehydrogenase gene (leu1 gene).
Usually, after carrying out homologous recombination, the obtained transformants are subjected to selection. The selection may, for example, be carried out as follows. Screening is carried out by a culture broth which can select transformants by the above-mentioned auxotrophic marker, and two or more colonies are selected among the obtained colonies. Then, after cultivating them separately in a liquid broth, the expression amount of gsf2 gene per cells in each liquid broth is measured so as to select a mutant showing higher expression amount. Further, the copy numbers of a vector and an expression cassette integrated into the chromosomes can be identified by subjecting the selected mutants to a genomic analysis using pulse-field gel electrophoresis.
The copy number of a vector integrated into the chromosomes can be adjusted to some extent by adjusting integration conditions, etc., but the integration efficiency and integration copy number also change due to the size (number of base pairs) and structure of the vector.
By deleting pvg1 gene or introducing a mutation which causes decrease or inactivation of the enzymatic activity of Pvg1 into pvg1 gene via a genetic engineering method, the enzymatic activity of Pvg1 of a S. pombe host can be decreased or inactivated. Deleting pvg1 gene per se from the chromosomes is preferred, since it ensures the complete inactivation of the enzymatic activity of Pvg1.
Deletion or inactivation of pvg1 gene can be carried out by publicly known methods. For example, the Latour system (Nucleic Acids Res. (2006) 34: e11, and WO2007/063919) can be used to delete pvg1 gene.
Further, the pvg1 gene may be inactivated by causing deletion, insertion, substitution or addition in a part of the nucleotide sequence of the pvg1 gene. The gene may be mutated by only one of the deletion, insertion, substitution and addition, or by two or more of them.
As a method for introducing the above-described mutation to a part of pvg1 gene, publicly known methods can be used. For example, a mutant screening method using mutagens (Koubo Bunshi Idengaku Jikken-Hou, 1996, Japan Scientific Societies Press), random mutations using PCR (polymerase chain reaction) (PCR Methods Appl., 1992, vol. 2, p. 28-33) and the like.
The S. pombe mutant of the present invention can also be obtained by an artificial mutation of a S. pombe which does not exhibit non-sexual flocculation. That is, it can be produced by subjecting a S. pombe which does not exhibit non-sexual flocculation to a mutation treatment, selecting cells exhibiting an increased Gsf activity as compared with a wild-type strain and having decreased or no Pvg1 enzymatic activity from the treated S. pombe, and further selecting ones resulting from dominant mutations in the increased Gsf activity and the decreased Pvg1 enzymatic activity, etc.
The mutation treatment of S. pombe may be carried out by using a mutagen such as EMS (ethyl methane sulfonate) or the like, or irradiating light of short wavelength such as ultraviolet rays or the like. Further, the selection of cells exhibiting acid-resistance non-sexual flocculation after subjecting S. pombe cells to a mutation treatment may be carried out in the presence of cations such as calcium ions.
The Gsf activity of a S. pombe mutant can be evaluated using, as an index, sedimentation rate. Thus, when S. pombe cells treated with mutagens are cultivated on a solid medium and colonies thereby formed are introduced into an appropriate solvent, the cells, which form colonies showing significantly faster sedimentation rate as compared with colonies of a wild-type strain (i.e. higher sedimentation rate), are evaluated as ones exhibit an increased Gsf activity as compared with wild-type strains. The colonies of a wild-type strain and the colonies of cells treated with mutagens can be introduced into solvents at almost the same time for comparing their sedimentation rates. The threshold value, determined by the preliminary measured sedimentation rate of a wild-type strain under a specific condition, may be compared with the sedimentation rate of the colonies of cells treated with mutagens. The solvents to be used for the sedimentation test of colonies are not particularly limited so long as they do not kill yeast cells, but are preferably buffers containing at least one cation selected from the group consisting of a calcium ion, a lithium ion, a manganese ion, a copper ion, and a zinc ion. For example, when the dried-cell concentration is 3.6 g/L, the cells having a sedimentation rate of at least 1.0 m/h in a calcium ion-containing lactate buffer solution (80 mM lactic acid, 100 mM calcium chloride, pH 6.0) can be selected as mutants exhibiting increased Gsf activity.
For example, an acid-resistance non-sexual flocculation strain can be obtained by the following operation. At first, mutation of S. pombe is induced by using EMS, and then mutated ones are isolated for cultivation. Then, cells collected after removing a culture supernatant are suspended in a lactic acid-sodium hydroxide buffer solution (80 mM lactic acid, 100 mM calcium chloride, pH 2.0) to a dried-cell concentration of 3.6 g/L, followed by measuring sedimentation rate to select a strain showing a sedimentation rate higher than 2.0 m/h as the acid-resistance non-sexual flocculation strain.
The mutant exhibiting increased Gsf activity can be selected by using, as an index, the expression amount of gsf2 gene. The expression amount of gsf2 gene may be measured by a measurement method generally used for gene expression analysis such as RT-PCR, Northern blotting using a labeled probe, or the like.
When the enzymatic activity of Pvg1 is decreased or inactivated, the amount of pyruvic acid contained in the cell surface tends to decrease significantly. Therefore, the enzymatic activity of Pvg1 of S. pombe can be evaluated using, as an index, the amount of pyruvic acid contained in the cell surface. That is, from S. pombe cells treated with mutagens, cells lacking pyruvic acid in their cell surfaces can be selected as a mutant exhibiting decreased or no Pvg1 enzymatic activity.
The mutant lacking pyruvic acid in its cell surface can by produced in accordance with a method of Andreishcheva et al., (The Journal of biological chemistry; 2004 Aug. 20, 279 (34):35644-55). At first, mutation of S. pombe cells is induced by using EMS, and the cells are cultivated for 48 hours in an appropriate liquid culture broth. Then, by using positively charged Q-sepharose, adhered cells are removed from the culture for collecting cells remained in the supernatant. After repeatedly carrying out the selection using Q-sepharose several times, the obtained culture supernatant was applied on a plate, followed by an isolation culture to obtain a mutant lacking pyruvic acid in its cell surface.
The mutant exhibiting decreased or no Pvg1 enzymatic activity can also be obtained by measuring the Pvg1 enzymatic activity of mutagen-treated cells. The Pvg1 enzymatic activity of S. pombe may be measured by a measurement method generally used for measuring enzymatic activities of other transferases such as a measurement method of using a labeled substrate, or the like.
The S. pombe mutant of the present invention may be prepared by combining a genetic engineering method and a mutation treatment. For example, a mutant in which the expression amount of gsf2 gene is increased by a mutation treatment may be subjected to a genetic engineering method to decrease or inactivate its Pvg1 enzymatic activity. Alternatively, a mutant in which its Pvg1 enzymatic activity is decreased or inactivated by a mutation treatment may be subjected to a genetic engineering method to increase the expression amount of gsf2 gene. Further, a mutant in which its Pvg1 enzymatic activity is decreased or inactivated by a genetic engineering method may be subjected to a mutation treatment to select a mutant in which the expression amount of gsf2 gene is increased.
The S. pombe mutant of the present invention may have a mutation in other genes so long as it maintains acid-resistance non-sexual flocculation, and further, it may contain a foreign structural gene in its chromosome or as an extrachromosomal gene.
The acid-resistance non-sexual flocculation of the S. pombe mutant of the present invention is not affected by the types of acids contained in a culture broth. That is, the S. pombe mutant is subject to non-sexual flocculation in all cases where the acid providing a culture broth pH range of from 2 to 5 is an organic acid such as lactic acid, citric acid, acetic acid, succinic acid, fumaric acid or malic acid, and where the acid is a mineral acid such hydrochloric acid or sulfuric acid.
As an index for the strength of non-sexual flocculation of the S. pombe mutant of the present invention, a sedimentation rate may, for example, be used. The sedimentation rate of yeast may, for example, be obtained by suspending yeast cells dispensed in a transparent container such as a test tube, letting them to stand still to start sedimentation, and dividing the distance between the liquid surface and the solid-liquid interface (interface between the sedimented yeast cells and the supernatant) by the time elapsed from the onset of sedimentation.
The S. pombe mutant of the present invention has a non-sexual flocculation property in a calcium ion-containing lactate buffer solution (80 mM lactic acid, 100 mM calcium chloride, pH 2.0). The sedimentation rate of the S. pombe mutant of the present invention in the calcium ion-containing lactate buffer solution is preferably at least 2.0 m/h, more preferably at least 4.0 m/h, further preferably at least 6.0 m/h, provided that the dried cell concentration is 3.6 g/L.
Further, the S. pombe mutant of the present invention has a non-sexual flocculation property in a calcium ion-containing lactate buffer solution (80 mM lactic acid, 100 mM calcium chloride, pH 4.0). The sedimentation rate of the S. pombe mutant of the present invention in the calcium ion-containing lactate buffer solution is preferably at least 2.0 m/h, more preferably at least 4.0 m/h, further preferably at least 8.0 m/h, provided that the dried cell concentration is 3.6 g/L.
When the sedimentation rate at pH 4.0 is at least 8.0 m/h, sufficient flocculation will be exhibited at a pH of lower than pH 4.
The acid-resistance non-sexual flocculation exhibited by the S. pombe mutant of the present invention may be dependent on at least one cation selected from the group consisting of a calcium ion, a lithium ion, a manganese ion, a copper ion, and a zinc ion. When the acid-resistance non-sexual flocculation is dependent on a calcium ion or the like, the aggregation of the S. pombe can be inhibited by adding a chelating agent such as EDTA to the culture broth.
The acid-resistance non-sexual flocculation exhibited by the S. pombe mutant of the present invention may be a property which is inhibited by galactose. When the acid-resistance non-sexual flocculation is inhibited by galactose, the aggregation of the S. pombe can be inhibited by adding galactose to the culture broth to a final concentration of at least 5 mM.
The S. pombe mutant of the present invention exhibits a strong non-sexual flocculation under an acidic condition (e.g. pH 2 to 5). Therefore, the S. pombe mutant is particularly suitable as a host of the expression system for synthesizing acidic proteins. Further, it is also suitable as a host of the expression system even in a case where the optimal pH for cultivation is lower than pH 5, considering the productivity of a desired protein.
When the below-described transformant of a S. pombe mutant of the present invention is prepared and the transformant is cultivated by a tank culture or the like, for the large scale production of β-glucosidase using the S. pombe mutant of the present invention as a host, even in a case where the pH of the culture broth is from 2 to 5 at the end of cultivation, cells can be aggregated without carrying out a solid-liquid separation treatment such as centrifugation or filtration, a neutralization treatment, etc., whereby the cells are easily separated from the culture broth. Further, the S. pombe mutant of the present invention may exhibit non-sexual flocculation under not only an acidic condition but also a weak acidic to alkaline (e.g. pH 5 to 10) condition.
[Transformant of S. pombe Mutant]
The transformant of a S. pombe mutant of the present invention is prepared by using the above-described S. pombe mutant, and has a structural gene sequence encoding a β-glucosidase derived from a filamentous fungus, and a promoter sequence and a terminator sequence for expressing the structural gene in a chromosome or as an extrachromosomal gene. Here, having the above-described expression cassette in a chromosome means that the expression cassette is integrated into at least one position of the chromosome of the yeast of the genus Schizosaccharomyces, and having as an extrachromosomal gene means that a plasmid having the expression cassette is contained in the yeast cell. From the viewpoint of easiness in subculture passage of the transformant, it is preferred to have the expression cassette in a chromosome.
The expression cassette is the same as one described in [S. pombe mutant], and is a combination of DNA necessary for expressing β-glucosidase, and contains a β-glucosidase structural gene, and a promoter and a terminator which function in a yeast of the genus Schizosaccharomyces. 
Further, since recovery and purification of a β-glucosidase become easier when the amount of a β-glucosidase secreted out of the cells of a yeast of the genus Schizosaccharomyces is large, it is preferred that a nucleotide sequence encoding a secretion signal sequence (structural gene of secretion signal) which functions in a yeast of the genus Schizosaccharomyces is located at the 5′ end side of the β-glucosidase structural gene. The 5′ end side of the β-glucosidase structural gene is a region upstream from the β-glucosidase structural gene, and is a position adjacent to the 5′ end of the g-glucosidase structural gene. Further, a nucleotide sequence encoding a number of amino acids in the N-terminal side, which does not affect the activity of β-glucosidase, may be removed and a gene encoding a secretion signal sequence may be introduced thereto.
The promoter and the terminator may be ones which function in a S. pombe mutant to direct expression of a β-glucosidase derived from a filamentous fungus. As the promoter which functions in a S. pombe mutant, those described in [S. pombe mutant] may be mentioned.
(β-glucosidase)
β-glucosidase (EC.3.2.1.21) is a generic name of an enzyme which specifically catalyzes hydrolysis of a β-D-glucopyranoside bond. Particularly, it is also called as cellobiase since it degrades cellobiose to glucose, and is widely found in bacteria, filamentous fungi, plants and animals. A plurality of genes encoding β-glucosidase is usually found in each of the species, and for example, existence of bgl1 to bgl7 in a filamentous fungus Aspergillus oryzae has been reported (Soy Protein Research, Japan, Vol. 12, pp. 78-83, 2009; and JP-A-2008-086310). Among them, bgl1 which encodes BGL1 is preferred from the viewpoint of its high activity, etc.
The structural gene of β-glucosidase contained in the transformant of a S. pombe mutant of the present invention is derived from a filamentous fungus.
The filamentous fungus is, among fungi, an eukaryotic microorganism composed of tubular cells called hyphae. As the filamentous fungus, a fungus of the genus Aspergillus, the genus Trichoderma, the genus Fusarium, the genus Penicillium, the genus Acremonium or the like may, for example, be mentioned. The structural gene of β-glucosidase of the present invention may be derived from any filamentous fungus so long as it produces β-glucosidase, but is preferably a β-glucosidase derived from a filamentous fungus of the genus Aspergillus from the viewpoint of its high enzymatic activity, etc. As the filamentous fungus of the genus Aspergillus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus aculeatus, Aspergillus niger, and Aspergillus pulverulentus may, for example, be mentioned. The gene encoding a β-glucosidase derived from Aspergillus aculeatus is preferred since it has high crystalline cellulose degradation ability and high yield of monosaccharide, and the gene encoding BGL1 (hereinafter also referred to as AaBGL1) derived from Aspergillus aculeatus is more preferred.
According to the doctoral dissertation of Dr. Reiichiro Sakamoto (Research on cellulase system of Aspergillus aculeatus No. F-50, Osaka Prefecture University, 1984), the wild type AaBGL1 purified from Aspergillus aculeatus has a molecular weight of about 133 kDa, an optimal pH of 4.0, and a stable pH range of from 3 to 7 (25° C., 24 hours).
The amino acid sequence of AaBGL1 is an amino acid sequence represented by SEQ ID NO: 1. The gene sequence encoding β-glucosidase of the present invention is preferably a gene sequence encoding a β-glucosidase comprised of the amino acid sequence represented by SEQ ID NO: 1. Further, it may be a gene sequence encoding a β-glucosidase comprised of the amino acid sequence represented by SEQ ID NO:1 having deletion, substitution or addition of from one to tens amino acids, preferably from one to few amino acids, more preferably from one to nine amino acids, and has a catalytic activity to hydrolyze a β-D-glucopyranoside bond.
The β-glucosidase comprised of the amino acid sequence represented by SEQ ID NO: 1 is one retains a catalytic activity to hydrolyze a β-D-glucopyranoside bond even in a case where deletion, substitution or addition of from one to tens amino acids is introduced into the sequence.
The above-described gene encoding a β-glucosidase derived from a filamentous fungus may be used as it is. However, to increase expression in a yeast of the genus Schizosaccharomyces, it is preferred to modify the above-described gene sequence by changing its codons to ones frequently used in a gene highly expressed in a yeast of the genus Schizosaccharomyces. 
In the present invention, as the vector for expressing β-glucosidase (hereinafter also referred to as bgl vector), a vector similar to the above-described gsf2 vector for expressing Gsf2 may be mentioned.
Further, the bgl vector preferably contains a secretion signal gene which functions in S. pombe. The secretion signal gene is located at the 5′ end side of the β-glucosidase structural gene. The secretion signal gene which functions in S. pombe is a gene encoding an amino acid sequence having a function of secreting the expressed foreign protein out of the host cell. A foreign protein to which the secretion signal is attached at its N-terminal is expressed from a foreign structural gene to which the secretion signal gene is bound. The secretion signal is removed from the foreign protein in the endoplasmic reticulum and the Golgi apparatus, etc. of the host cell, and then, the foreign protein detached from the secretion signal is secreted out of the host cell. The secretion signal gene (and the secretion signal) should be capable of functioning in a S. pombe mutant. As the secretion signal gene capable of functioning in a S. pombe mutant, the secretion signal genes described in WO1996/23890 may be used.
In the present invention, the structural gene of the secretion signal is introduced at the 5′ end side of the β-glucosidase structural gene, whereby it becomes possible to express a β-glucosidase to which the secretion signal is attached at its N-terminal, and then secrete the β-glucosidase out of the cells of yeast of the genus Schizosaccharomyces. As the secretion signal capable of functioning in a yeast of the genus Schizosaccharomyces, P3 signal described in WO1996/23890 is particularly preferred.
By using the above-described bgl vector, a S. pombe mutant, as a host, is transformed. The introduction of the β-glucosidase structural gene into the S. pombe mutant may be carried out in the same manner as in the introduction of gsf2 gene. Further, the selection of a transformant may be carried out in the same manner.
As the culture broth for cultivating the S. pombe mutant of the present invention, a publicly known culture broth for yeasts may be used so long as it contains carbon sources, nitrogen sources, inorganic salts and the like which yeast of the genus Schizosaccharomyces can use, and yeast of the genus Schizosaccharomyces can grow in it efficiently. The culture broth may be natural or synthetic.
As the carbon sources, saccharides such as glucose, fructose and sucrose may, for example, be mentioned.
As the nitrogen sources, inorganic acids or inorganic ammonium salts such as ammonia, ammonium chloride, and ammonium acetate, peptone and casamino acid may, for example, be mentioned.
As inorganic salts, magnesium phosphate, magnesium sulfate and sodium chloride may, for example, be mentioned.
Cultivation may be carried out by using a publicly known cultivation method for yeasts such as a shaking cultivation, a stirring cultivation or the like.
The cultivation temperature is preferably from 23 to 37° C. Further, the cultivation time may be set appropriately.
Cultivation may be carried by batch culture, fed-batch culture or continuous culture.
In the case of using, as the transformant of a S. pombe mutant of the present invention, a transformant having a β-glucosidase structural gene to which the secretion signal gene is bound, β-glucosidase is secreted into the culture broth. Then, when the transformant is cultivated in a tank culture or the like for the large scale production of β-glucosidase, since the transformant exhibits non-sexual flocculation even in a case where the pH of the culture broth is from 2 to 5 at the end of cultivation, cells can be aggregated without carrying out a solid-liquid separation treatment such as centrifugation or filtration, a neutralization treatment, etc., whereby the cells are easily separated from the culture broth.
Further, in the case of using, as the transformant of a S. pombe mutant of the present invention, a transformant having a β-glucosidase structural gene to which the secretion signal gene is not bound, a publicly known protein separation method may be used for separating a β-glucosidase.
For example, after cultivation, sedimented cells are separated from the culture broth and the cells are disrupted to obtain a cell lysate containing a β-glucosidase, and then the β-glucosidase is recovered by using a publicly known protein isolation method such as sating-out, column purification, chromatography or immunoprecipitation. Next, the second embodiment of the present invention will be described.
[Cloning Vector]
The cloning vector of the second embodiment of the present invention is a cloning vector for producing an expression vector to be introduced into a yeast of the genus Schizosaccharomyces for the expression of a foreign protein, and is characterized by having hsp9 promoter or ihc1 promoter of the yeast of the genus Schizosaccharomyces as a promoter which controls the expression of the foreign protein. Further, hereinafter, the cloning vector of the second embodiment of the present invention may also be referred to as the cloning vector of the present invention.
<hsp9 Promoter>
The hsp9 gene of a yeast of the genus Schizosaccharomyces is a gene encoding Hsp9 which is a kind of a heat shock protein (hsp) of a yeast of the genus Schizosaccharomyces. The systematic name of hsp9 gene registered in a gene sequence database of S. pombe (S. pombe Gene DB; HyperText Markup Language://WorldWideWeb.genedb.org/genedb/pombe/) is SPAP8A3.04c,
Heat shock protein (hsp) is a generic name for proteins which are induced to be synthesized when a cell or an organism is suddenly exposed to a temperature 5 to 10° C. higher than its physiological temperature (heat shock) and function as a chaperon to protect proteins from thermal denaturation or aggregation. In vivo synthesis of heat shock proteins is induced by various chemical substances such as electron transport chain inhibitors, transition metals, SH reagents and ethanol as well as by heat shock.
Therefore, in the transformant prepared by introducing an expression vector having a hsp9 gene promoter, it is possible to control the expression of a foreign gene, in the same manner as in the expression of Hsp9 protein, by heat shock or stimulation with various chemical substances.
The expression efficiency of hsp9 promoter in a yeast of the genus Schizosaccharomyces is significantly high. Accordingly, by using this promoter, it becomes possible to prepare an expression vector which can produce an unexpectedly large amount of a foreign protein from a transformant.
The hsp9 promoter may be a promoter of hsp9 gene contained in a yeast of the genus Schizosaccharomyces, and may also be a promoter derived from any yeast of the genus Schizosaccharomyces, but is preferably a hsp9 promoter of S. pombe which is more widely used. The hsp9 promoter of S. pombe is a region contained in 1 to 400 bp upstream from the 5′ end (A of initiation codon ATG) of a hsp9 gene ORF (SEQ ID NO:6).
As yeasts of the genus Schizosaccharomyces, other than S. pombe, which have hsp9 promoter, Schizosaccharomyces japonicus and Schizosaccharomyces octosporus may, for example, be mentioned. Further, the hsp9 promoter used for a cloning vector may be one derived from the same species as, or one derived from a species different from, the yeast of the genus Schizosaccharomyces to be introduced with an expression vector prepared from the cloning vector.
The hsp9 promoter is a region comprised of a nucleotide sequence identical to the promoter endogenous to a wild-type yeast of the genus Schizosaccharomyces (wild-type hsp9 promoter), or a region which has a promoter activity similar to the wild-type hsp9 promoter and is comprised of the nucleotide sequence having deletion, substitution or addition of at least one amino acid, preferably from one to tens amino acids, more preferably from one to dozen amino acids, further preferably from one to nine amino acids, even further preferably from one to few amino acids.
Further, the hsp9 promoter to be used for the cloning vector of the present invention may be a region which has a promoter activity similar to the wild-type hsp9 promoter and is comprised of an amino acid sequence having a homology to a nucleotide sequence identical to the wild-type hsp9 promoter of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%.
The hsp9 promoter of S. pombe is a region contained in 1 to 400 bp upstream from the 5′ end (A of initiation codon ATG) of a hsp9 gene ORF. The nucleotide sequence of the region is shown as SEQ ID NO:6. That is, the cloning vector of the present invention preferably contains a region comprised of a nucleotide sequence represented by SEQ ID NO:6. Further, a region which has a promoter activity similar to the wild-type hsp9 promoter and is comprised of a nucleotide sequence represented by SEQ ID NO:6 having deletion, substitution or addition of at least one amino acid, preferably from one to tens amino acids, more preferably from one to dozen amino acids, further preferably from one to nine amino acids, even further preferably from one to few amino acids, or a nucleotide sequence having a homology to a sequence represented by SEQ ID NO:6 of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, may also be used suitably as the hsp9 promoter for the cloning vector of the present invention.
<ihc1 Promoter>
ihc1 gene is a gene encoding Ihc1 which is a protein having a molecular weight of 15,400. ihc1 gene is widely conserved among fungi including a yeast of the genus Schizosaccharomyces. 
The expression of Ihc1 protein is suppressed in a low-cell-density state such as the beginning of cell growth, and is induced in a high-cell-density state. The expression of the Ihc1 protein is controlled by the promoter of ihc1 gene. Therefore, the induction of expression by ihc1 promoter is suppressed in a low-cell-density state such as the beginning of cell growth, and the expression is highly inducible in a high-cell-density state. Accordingly, by using this promoter, it becomes possible to prepare an expression vector which enables, in a transformant of a yeast of the genus Schizosaccharomyces, the adjustment of the expression of a foreign protein in a cell-density dependent manner.
The ihc1 promoter may be a promoter of ihc1 gene contained in a yeast of the genus Schizosaccharomyces, and may also be a promoter derived from any yeast of the genus Schizosaccharomyces, but is preferably ihc1 promoter of S. pombe which is more widely used.
The ihc1 gene of S. pombe is publicly known, and the systematic name of ihc1 gene registered in a gene sequence database of S. pombe (S. pombe Gene DB; HyperText Markup Language://WorldWideWeb.genedb.org/genedb/pombe/) is SPAC22G7.11c. The ihc1 promoter is a region contained in 1 to 501 bp upstream from the 5′ end (A of initiation codon ATG) of an ihc1 gene ORF (SEQ ID NO:9).
The ihc1 promoter to be used for the cloning vector of the present invention may be a promoter of ihc1 gene contained in a yeast of the genus Schizosaccharomyces, and may also be a promoter derived from any yeast of the genus Schizosaccharomyces. As the yeast of the genus Schizosaccharomyces, S. pombe, Schizosaccharomyces japonicus and Schizosaccharomyces octosporus may, for example, be mentioned. Further, the ihc1 promoter used for a cloning vector may be one derived from the same species as, or one derived from a species different from, the yeast of the genus Schizosaccharomyces to be introduced with an expression vector prepared from the cloning vector. In the present invention, it is preferred to use ihc1 promoter of S. pombe which is more widely used.
The ihc1 promoter is a region comprised of a nucleotide sequence identical to the promoter endogenous to a wild-type yeast of the genus Schizosaccharomyces (wild-type ihc1 promoter), or a region which has a promoter activity similar to the wild-type ihc1 promoter and is comprised of the nucleotide sequence having deletion, substitution or addition of at least one amino acid, preferably from one to tens amino acids, more preferably from one to dozen amino acids, further preferably from one to nine amino acids, even further preferably from one to few amino acids.
Further, the ihc1 promoter to be used for the cloning vector of the present invention may be a region which has a promoter activity similar to the wild-type ihc1 promoter and is comprised of an amino acid sequence having a homology to a nucleotide sequence identical to the wild-type ihc1 promoter of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%.
The ihc1 promoter of S. pombe is a region contained in 1 to 501 bp upstream from the 5′ end (A of initiation codon ATG) of an ihc1 gene ORF (SEQ ID NO:9). The nucleotide sequence of the region is shown as SEQ ID NO:9. That is, the cloning vector of the present invention preferably contains a region comprised of a nucleotide sequence represented by SEQ ID NO:9. Further, a region which has a promoter activity similar to the wild-type hsp1 promoter and is comprised of a nucleotide sequence represented by SEQ ID NO:9 having deletion, substitution or addition of at least one amino acid, preferably from one to tens amino acids, more preferably from one to dozen amino acids, further preferably from one to nine amino acids, even further preferably from one to few amino acids, or a nucleotide sequence having a homology to a sequence represented by SEQ ID NO:9 of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, may also be used suitably as the ihc1 promoter for the cloning vector of the present invention.
<Cloning Vector>
The cloning vector of the present invention has, in addition to the hsp9 promoter or the ihc1 promoter, a cloning site for introducing a foreign structural gene which is located downstream from the promoter and is governed by the promoter, and a terminator capable of functioning in a yeast of the genus Schizosaccharomyces. 
The cloning site contained in the cloning vector is a restriction enzyme recognition site exists only in the cloning site of the cloning vector. The cloning site contained in the cloning vector of the present invention may have only one restriction enzyme recognition site, or may be a multiple cloning site having at least two restriction enzyme recognition sites. As the multiple cloning site, a multiple cloning site contained in publicly known multiple cloning vectors can be used as it is, and one prepared by appropriately modifying a publicly known multiple cloning site can also be used. In addition, the cloning vector of the present invention may have a stop codon at a downstream end region inside of the cloning site or at the downstream of the cloning site.
As the terminator which functions in a yeast of the genus Schizosaccharomyces, a terminator endogenous to the yeast of the genus Schizosaccharomyces or a terminator exogenous to the yeasts of the genus Schizosaccharomyces can be used. Further, two or more types of terminators may be present in the vector. As the terminator endogenous to a yeast of the genus Schizosaccharomyces, an inv1 gene terminator of a yeast of the genus Schizosaccharomyces may, for example, be mentioned. Further, as the terminator exogenous to a yeast of the genus Schizosaccharomyces, the terminator derived from human disclosed in Patent Document 2, 4 or 10 may, for example, be mentioned, and human lipocortin-1 terminator is preferred.
The cloning vector of the present invention preferably contains a 5′-untranslation region located downstream from the promoter and upstream from the cloning site, and preferably contains a 3′-untranslation region located downstream from the cloning site. Further, the cloning vector of the present invention preferably contains, at the cloning site, a marker for discriminating it from an expression vector having a foreign structural gene introduced therein. As the marker, a drug resistance gene capable of functioning in E. coli such as an ampicillin resistance gene may, for example, be mentioned.
Further, the cloning vector of the present invention preferably contains a marker for selecting a transformant. As the marker, an auxotrophic complementation marker such as ura4 gene and isopropyl malate dehydrogenase gene (leu1 gene) may, for example, be mentioned.
The cloning vector of the present invention may further contain, in addition to a region which constitutes an expression cassette when a foreign structural gene is introduced into the cloning site, a DNA region necessary for producing a transformant. For example, in the case of introducing an expression cassette into a chromosome, a recombination region is preferably contained therein. The recombination region described in the first embodiment of the present invention, which is used for introducing the expression cassette of gsf2 vector into a chromosome, may be used as it is for introducing an expression cassette containing a foreign structural gene (not limited to gsf2 gene). Even in the case of an expression vector containing a foreign structural gene other than gsf2 gene, for introducing the expression cassette into a chromosome, the genetic engineering method described in the first embodiment of the present invention may be used as it is.
In the case of preparing a transformant in which the expression cassette of the expression vector produced from the cloning vector of the present invention is maintained in host cells as an extrachromosomal gene, the vector of the present invention is preferably a plasmid which contains a sequence required for replication in yeast of the genus Schizosaccharomyces, i.e. Autonomously Replicating Sequence (ARS). Further, in the case of integrating the expression cassette into a chromosome, ARS is preferably eliminated from the expression vector before its introduction into a host.
The cloning vector of the present invention can be produced by replacing a promoter region, which is contained in a publicly known cloning vector to be used for producing the expression vector for expressing a foreign gene in a host, with a hsp9 promoter or ihc1 promoter. For example, it can be produced by replacing the promoter region of a multiple cloning vector, which is disclosed in JP-A-H7-163373, JP-A-H10-234375, JP-A-H11-192094, JP-A-2000-136199 or the like, with a hsp9 promoter or ihc1 promoter.
As specific methods for constructing the cloning vector of the present invention, publicly known methods can be used. For example, an operation method described in the article [J. Sambrook et al., “Molecular Cloning 2nd ed.”, Cold Spring Harbor Laboratory Press (1989)]. In addition, it may be constructed by an enzymatic amplification method using PCR, a chemical synthesis, or the like.
[Expression Vector and its Production Method]
The expression vector of the second embodiment of the present invention can be produced by introducing a foreign structural gene into a cloning site of the cloning vector of the present invention. The introduction of the foreign structural gene into the cloning site may be carried out by using publicly known methods, like the production of the cloning vector.
The foreign structural gene introduced in the expression vector of the present invention is not particularly limited so long as it is a structural gene encoding a protein, and may be a gene homologous to a gene endogenous to the yeast of the genus Schizosaccharomyces host or a structural gene derived from a heterologous organism. From the yeast of the genus Schizosaccharomyces transformant obtained by using an expression vector containing a structural gene (e.g. the above-mentioned gsf2 gene) encoding an endogenous protein of a yeast of the genus Schizosaccharomyces, a large amount of the endogenous protein can be produced. Further, from the yeast of the genus Schizosaccharomyces transformant obtained by using an expression vector containing a structural gene derived from a heterologous organism, a large amount of heterologous proteins can be produced.
The protein encoded by a foreign structural gene introduced into the expression vector of the present invention is preferably a heterologous protein, more preferably a protein produced by multicellular organisms such as animals and plants, especially a protein produced by a mammal (including humans). Such a protein is rarely obtained with high activity if a prokaryotic host microorganism such as E. coli is used for its production, and its production efficiency is generally low if an animal cell such as CHO cell is used as a host. These problems can be solved by using the expression vector of the present invention and employing a heterologous protein expression system in which a yeast of the genus Schizosaccharomyces is used as a host.
The foreign structural gene introduced in the expression vector of the present invention may be a wild-type structural gene, a gene prepared by modifying a wild-type structural gene, or an artificially synthesized gene, so long as it encodes a protein. As a non-wild-type structural gene, a gene encoding a chimeric protein in which two or more wild-type proteins are fused one another and a gene encoding a protein in which an additional peptide or the like is bound to the N-terminal or C-terminal of a wild-type protein may, for example, be mentioned. As the additional peptide, a signal such as a secretion signal, an organelle localization signal or the like, and a tag such as His-tag or FLAG-tag may, for example, be mentioned. The signal should be a signal which functions in a yeast of the genus Schizosaccharomyces. The secretion signal is a peptide introduced at the N-terminal and having a function of secreting the expressed protein out of the host cell. As the secretion signal which functions in a yeast of the genus Schizosaccharomyces, P3 signal described in WO1996/23890 is particularly preferred.
[Transformant and its Production Method]
The transformant of the second embodiment of the present invention is characterized by containing the above-described expression vector of the second embodiment of the present invention. The transformant of the second embodiment of the present invention is produced by introducing the above-described expression vector into a yeast of the genus Schizosaccharomyces. 
The host for the transformant of the second embodiment of the present invention is a yeast of the genus Schizosaccharomyces. It may be a wild-type or a mutant-type in which a specific gene is deleted or inactivated depending on application. For deletion or inactivation of a specific gene, publicly known methods can be used. Specifically, the Latour system (Nucleic Acids Res. (2006) 34: e11, and WO2007/063919) can be used to delete the gene. Further, the gene can be inactivated by mutating the gene at a certain position by mutant screening using mutagens (Koubo Bunshi Idengaku Jikken-Hou, 1996, Japan Scientific Societies Press), random mutations using PCR (PCR Methods Appl., 1992, vol. 2, p. 28-33) and the like. As the yeast of the genus Schizosaccharomyces host in which a specific gene is deleted or inactivated, ones disclosed in WO2002/101038, WO2007/015470, etc. may be used.
Further, the host is preferably a yeast of the genus Schizosaccharomyces having a marker for selecting a transformant. For example, it is preferred to use a host which essentially requires a specific nutrient factor for growth due to deletion of a certain gene. When preparing a transformant by using a vector containing a target gene sequence, a transformant lacking the auxotrophy of the host can be obtained by using a vector carrying the deleted gene (auxotrophic complementation marker). It is possible to select the transformant by using the difference in auxotrophy between the host and the transformant. As the auxotrophic complementation marker, ura4 gene (auxotrophic complementation marker) and isopropyl malate dehydrogenase gene (leu1 gene) may, for example, be mentioned.
As the yeast of the genus Schizosaccharomyces host, one belongs to the above-mentioned species may be used. Among the above-described yeasts of the genus Schizosaccharomyces, S. pombe is preferred in view of the availability of various useful mutant strains. The S. pombe strain to be used in the present invention may, for example, be ATCC38399 (leu1-32h−) or ATCC38436 (ura4-294h−), which is available from the American Type Culture Collection.
Further, even in the case of introducing an expression vector containing a foreign structural gene other than β-glucosidase gene, the S. pombe mutant of the first embodiment of the present invention may be used as a host.
The yeast of the genus Schizosaccharomyces host is transformed by using the above-described expression vector, and as the transformation method, any known transformation method for a yeast of the genus Schizosaccharomyces may be used. Such a transformation method may, for example, be a conventional method like a lithium acetate method [K. Okazaki et al., Nucleic Acids Res., 18, 6485-6489 (1990)], electroporation method, spheroplast method, glass-beads method, or the like., and a method disclosed in JP-A-2005-198612. Further, a commercially available yeast transformation kit may be used.
After transformation, the resulting transformants are usually subjected to selection. The selection may, for example, be carried out as follows. Screening is carried out by a culture broth which can select transformants by the above-mentioned auxotrophic marker, and two or more colonies are selected among the obtained colonies. In addition, the copy numbers of a vector and an expression cassette integrated into the chromosomes can be identified by subjecting the selected mutants to a genomic analysis using pulse-field gel electrophoresis.
(Cultivation Method)
The transformant of the second embodiment of the present invention may be cultivated in the same manner as a natural yeast of the genus Schizosaccharomyces. As the cultivation method, ones described in the first embodiment of the present invention may be mentioned. Specifically, a nutrient medium such as YPD medium (M. D. Rose et al., “Methods In Yeast Genetics”, Cold Spring Harbor Laboratory Press (1990)), a minimal medium such as MB Medium (K. Okazaki et al., Nucleic Acids Res., vol. 18, p. 6485-6489 (1990)) and the like may be used.
Publicly known yeast cultivation methods including a shaking cultivation and a stirring cultivation may, for example, be used.
Further, the cultivation temperature is preferably from 23 to 37° C. Further, the cultivation time may be set appropriately.
Cultivation may be carried by batch culture, fed-batch culture or continuous culture.
[Method for Producing a Foreign Protein]
The method for producing a protein of the second embodiment of the present invention is characterized by cultivating the above-described transformant of the second embodiment of the present invention and, from a cell or a culture supernatant thereby obtained, recovering a protein encoded by the above-described foreign structural gene.
The cultivation conditions can be set appropriately taking into consideration the type, etc. of a foreign protein of interest to be produced. For example, at a temperature of from 16 to 42° C., preferably from 25 to 37° C., and a cultivation time of from 8 to 168 hours, preferably from 48 to 96 hours. Either shaking culture or static culture can be employed, and stirring or aeration may be applied if necessary.
When the transformant of the second embodiment of the present invention is a transformant prepared by introducing an expression vector containing a hsp9 promoter, after cultivating the transformant under a condition where an inductive stimulus such as heat stress is applied thereto, the hsp9 promoter is activated by the stress and the transcription of a foreign structural gene governed by the promoter is promoted, whereby the foreign structural protein is expressed. In the case of cultivating under such an inductive stimulus condition, as compared with the case of cultivating under a normal condition, the growth amount of a yeast of the genus Schizosaccharomyces is generally low. Therefore, the cultivation is carried out under a normal condition at the beginning of cultivation, and then an inductive stimulus such as heat stress is applied thereto at the time when the concentration of cells in a culture broth is increased to a certain level. Since the amount of cells is increased by the initial cultivation stage, the cultivation system as a whole produces a large amount of heterologous proteins. Here, heat stress is one of available inductive stimuli, and other inductive stimuli may be used as long as their effects are verified as mentioned above. The inductive stimulus is preferably heat, or addition of cadmium, an osmotic pressure increasing agent, hydrogen peroxide, ethanol or the like.
In the case of heat, the temperature can be increased to the maximum survival temperature of a yeast of the genus Schizosaccharomyces. Therefore, the temperature for applying heat stress is a temperature of, preferably from 2 to 20° C., more preferably from 3 to 12° C., most preferably from 4 to 6° C., higher than the original cultivation temperature, and is from 15 to 55° C., preferably from 25 to 45° C., more preferably from 30 to 40° C. The heat stress application time is not particularly restricted, but its effect can be confirmed after at least several minutes, and is preferably from 1 to 29 hours, more preferably from 1 to 15 hours.
In the case of cadmium addition, it is added in the form of cadmium ions. The final cadmium concentration is from 0.1 to 1.5 mM, preferably from 0.5 to 1.0 mM. The cultivation time is preferably at most 5 hours, particularly preferably at most 3 hours.
In the case of an osmotic pressure increasing agent, an osmotic pressure increasing agent such as a high concentration electrolyte or sorbitol is added to increase the osmotic pressure. When using potassium chloride at a high concentration, the final potassium concentration is from 0.1 to 2.0 M, preferably from 0.5 to 1.5 M. The addition time is not particularly limited, but is preferably from 1 to 12 hours, more preferably from 1 to 10 hours.
In the case of hydrogen peroxide, its final concentration is from 0.1 to 1.5 mM, preferably from 0.5 to 1.0 mM. The cultivation time is not particularly limited, but is preferably from 1 to 15 hours, more preferably from 1 to 12 hours.
In the case of ethanol, its final concentration is from 5 to 20 V/V %, preferably from 5 to 15 V/V %. The cultivation time is not particularly limited, but is preferably from 1 to 20 hours, particularly preferably from 1 to 15 hours.
The above-mentioned conditions may be applied alone or in combination of two or more of them. The effect of such a combination can be ascertained easily by comparing the expression amounts.
When the transformant of the second embodiment of the present invention is a transformant prepared by introducing an expression vector containing an ihc1 promoter, after cultivating the transformant in a low-cell-density state such as the beginning of cell growth, no expression or significantly low expression of a foreign protein is observed. That is, in a low-cell-density state, the transformant can grow with no (or little) burden of expressing a foreign protein and can grow more efficiently as compared with a case of growing with the burden, whereby the amount of cells can be increased efficiently. On the other hand, as the cell density increases, the induction of the expression proceeds. As a result, the foreign protein can be produced in a large amount.
At the end of cultivation, cells are ruptured sonically or mechanically to obtain a cell extract containing the foreign protein of interest, whereby the foreign protein can be isolated and purified from the cell extract. Further, in a case where the foreign protein is secreted out of the cells, the foreign protein can be isolated and purified from the culture supernatant. As the isolation and purification method for recovering the produced protein, publicly known methods including a method utilizing difference in solubility such as salting out or solvent precipitation, a method utilizing difference in molecular weight such as dialysis, ultrafiltration or gel electrophoresis, a method utilizing difference in electric charge such as ion-exchange chromatography, a method utilizing specific affinity such as affinity chromatography, a method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, and a method utilizing difference in isoelectric point such as isoelectric focusing may, for example, be mentioned.
The isolated and purified protein can be identified by a publicly known method such as western blotting or an activity measurement method. The structure of the purified protein can be identified by amino acid analysis, amino-terminal analysis, primary structure analysis and the like.
EXAMPLES
Now, the present invention will be described in further detail with reference to Examples and Comparative Examples. However, it should be understood that the present invention is by no means thereby restricted.
[Test Example 1] Expression Vector Preparation 1
A gene sequence was designed based on the peptide sequence of AaBGL1, by replacing the codons with codons highly expressed in S. pombe (SEQ ID NO: 2. Hereinafter referred to as AaBGL1 gene). The recognition sequences for KpnI and BspHI were added upstream of the initiation codon. The recognition sequences for XbaI and SacI were added downstream of the stop codon. A plasmid containing these sequences (synthesized by Geneart AG, Regensburg, Germany) was digested with restriction enzymes BspHI and XbaI.
On the other hand, separately therefrom, pSL6lacZ was digested with restriction enzymes AarI and XbaI, and then treated with an alkaline phosphatase. Thereafter, gel electrophoresis was carried out on an agarose gel to isolate the digested fragment of vector pSL6 and the digested fragment of AaBGL1 gene from the agarose gel, and then these fragments were ligated to each other. The ligated product was introduced into E. coli DH5α (Takara Bio, Inc.) to obtain a transformant. From the obtained transformant, a vector was prepared to obtain expression vector pSL6AaBGL1 (FIG. 1, refer to SEQ ID NO: 3). The obtained expression vector was confirmed to be a desired vector by restriction enzyme mapping.
Further, to prepare AaBGL1 to which secretion signal P3 is attached at the N-terminal, a fragment of AaBGL1 gene was amplified by PCR method with In-fusion primers and pSL6AaBGL1 as template. On the other hand, pSL6P3lacZ was digested with restriction enzymes AR and XbaI. The digested fragments and the PCR-amplified product of the AaBGL1 gene fragment were circularized by In-fusion method, and then introduced into E. coli DH5α (Takara Bio, Inc.) to obtain a transformant. From the obtained transformant, a vector was prepared to obtain a desired expression vector pSL6P3AaBGL1 (FIG. 2, refer to SEQ ID NO: 4). The obtained expression vector was confirmed to be a desired vector by restriction enzyme mapping and partial nucleotide sequencing.
[Test Example 2] Expression Vector Preparation 2
Further, to prepare AaBGL1 expression vector using a hsp9 promoter, a fragment of pSL6P3AaBGL1 gene was amplified by PCR method with In-fusion primers and pSL6P3AaBGL1 as template. On the other hand, the below-described pSL14lacZ containing a hsp9 promoter was digested with restriction enzymes AarI and XbaI. The digested fragments and the PCR-amplified product of the P3AaBGL1 gene fragment were circularized by In-fusion method, and then introduced into E. coli DH5α to obtain a transformant. From the obtained transformant, a vector was prepared to obtain a desired expression vector pSL14P3AaBGL1 (FIG. 3, refer to SEQ ID NO: 5). The obtained expression vector was confirmed to be a desired vector by restriction enzyme mapping and partial nucleotide sequencing.
<Preparation of Multiple Cloning Vector>
The promoter region (“inv1 pro.” in FIG. 24) of multiple cloning vector pSL9 (FIG. 24, 7,038 bp) was replaced with a hsp9 promoter of S. pombe (SEQ ID NO: 6) to prepare multiple cloning vector pSL14 (FIG. 25, 6,282 bp).
Specifically, at first, a region 1 to 400 bp (SEQ ID NO: 6) upstream from the 5′ end (A of initiation codon ATG) of the ORF of S. pombe hsp9 gene was amplified by using genomic DNA derived from a wild-type strain of S. pombe (ARC032 strain, corresponds to ATCC38366, 972h−) as a template, a forward primer comprising the restriction enzyme recognition site for SacI at the 5′ end, and a reverse primer comprising the restriction enzyme recognition site for PciI at the 5′ end, thereby to obtain a fragment having the restriction enzyme recognition site for SacI at the 5′ end and the restriction enzyme recognition site for PciI at the 3′ end.
The promoter portion of pSL9 was subjected to double digestion with restriction enzymes AarI and SalI, followed by ligation for transforming E. coli DH5α. After extracting a plasmid, its nucleotide sequences was identified. As a result, the obtained plasmid was found to contain a promoter region having a nucleotide sequence represented by SEQ ID NO: 6, while the sequence has one additional adenine at its 3′ end. Thus obtained plasmid was named as pSL14.
Then, the region including an ampicillin resistance gene and pBR322ori of pSL14 was replaced with a region including a kanamycin resistance gene and pUCori, and a structural gene encoding lacZ′ was integrated into the cloning site, whereby multiple cloning vector pSL14lacZ (FIG. 26, 6,657 bp, refer to SEQ ID NO: 7).
[Test Example 3] Preparation of a Transformant (Transformation of Fission Yeast)
As the host cell, a leucine-auxotrophic strain of S. pombe (Genotype: h−, leu1-32, provided from professor Yuichi lino, Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo) (ATCC38399) was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of the above-obtained vector pSL14P3AaBGL1 digested by restriction enzyme SwaI was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium. Three days after cultivation, a transformant (AaBGL1 expression strain) was obtained. Thus obtained transformant was named as ASP3660 strain (hereinafter also referred to as normal strain).
[Test Example 4] Preparation of ΔPvg1 Strain (Pvg1 Gene Deletion Strain)
As the host cell, a uracil-auxotrophic strain of S. pombe (ARC010 strain, Genotype: h−leu1-32 ura4-D18, provided from professor Yuichi lino, Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo) was transformed in accordance with the Latour system (Nucleic Acids Res. (2006) 34: e11, and WO2007/063919) to prepare Δpvg1 strain in which pvg1 gene was deleted. Thus obtained mutant was cultivated and subjected to genomic analysis with pulse-field gel electrophoresis, thereby to confirm that pvg1 gene was deleted.
The preparation of a deletion fragment was carried out by a PCR method using whole genomic DNA obtained with DNeasy (manufactured by QIAGEN) from a wild-type strain ARC032 of S. pombe (Genotype: h−, provided from professor Yuichi lino, Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo) as the template.
Specifically, a deletion fragment was divided into UP region, OL region and DN region, and DNA fragments of these regions were prepared by a PCR method of using KOD-Dash (manufactured by Toyobo Co. Ltd.), and then full-length deletion fragments were prepared by a similar PCR method using the fragments as templates.
[Test Example 5] Preparation of Δpvg1gsf2+ Strain (Pvg1 Gene Deletion+Gsf2 Expression Increase Strain)
The above-described Δpvg1 strain was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1 M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of the below-described gene fragment (SEQ ID NO: 8) containing an ihc1 promoter and gsf2 gene was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium containing leucine. Three days after cultivation, a transformant (pvg1 gene deletion+gsf2 expression increase strain) was obtained. FOA treatment was carried out to make it auxotrophic for uracil again. Thus obtained mutant was named as IGF799 strain.
The above-described gene fragment containing an ihc1 promoter and gsf2 gene was prepared as follows. At first, a sequence containing an ihc1 promoter (SEQ ID NO: 9), a gsf2 promoter sequence and a Ura4 sequence at the 5′ end side of the ihc1 promoter, and a gsf2-ORF at the 3′ end side was prepared as a template, and then amplified by PCR to obtain the above-mentioned gene fragment.
[Test Example 6] Preparation of a Transformant (Transformation of a Non-Sexual Flocculation Fission Yeast)
As the host cell, the above-described IGF799 strain of S. pombe which exhibits non-sexual flocculation was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1 M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of the above-obtained vector pSL14P3AaBGL1 digested by restriction enzyme SwaI was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium containing uracil. Three days after cultivation, a transformant (AaBGL1 expression strain) was obtained. Thus obtained transformant was named as ASP4106 strain.
[Test Example 7] Preparation of a Transformant (Uracil-Auxotrophy Complementation)
The above-prepared ASP4106 strain was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1 M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of pUC19-ura4 (FIG. 4, refer to SEQ ID NO: 10) vector digested by restriction enzyme Bsiwl was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium. In FIG. 4, the sequences of A and B indicate the recombination regions of the vector. Three days after cultivation, a transformant was obtained. Thus obtained transformant lacking the uracil-auxotrophy of ASP4106 strain was named as ASP4150 strain (hereinafter also referred to as flocculation strain).
[Test Example 8] Batch Culture of a Transformant
The above-obtained AaBGL1 expression strains (normal strain and flocculation strain) were cultivated in YES medium by a test tube for 24 hours at 32° C. 2 ml of the culture broth was transferred to 50 ml of YPD medium (1% of yeast extract, 2% of peptone, and 2% of glucose), and then cultivated by a 500 ml Erlenmeyer flask for 48 hours at 32° C.
To evaluate the cell growth of the transformant, the absorbance of a culture broth at OD660 nm was measured by a spectrophotometer (Spectrophotometer U-1500). In a case where the concentration of the culture broth is high, dilution with RO (Reverse Osmosis) water was carried out for the measurement. In the case of measuring the OD660 of the flocculation strain, before the measurement, suspension with 100 mM to 500 mM EDTA was carried out for deflocculation.
The results of the cell growth are shown in FIG. 5. Comparing the normal strain and the flocculation strain, the time-course changes in the OD660 values during the cultivation were generally similar, and the final OD660 values of the normal strain and the flocculation strain were found to be 23.4 and 20.3, respectively.
The concentration of residual glucose or a metabolite thereof, ethanol, in the culture broth was measured by a biosensor BF5. The collected culture broth was transferred to an Eppendorf tube, and a culture supernatant was obtained by centrifugation using a high-speed microcentrifuge. 300 μl of the culture supernatant was transferred to a BF5 sampling cup, and then the cup was placed in BF5 autosampler which had already been adjusted for the measurement.
BF5 was operated under the following biosensor operation conditions for the analysis.
Equipment: Biosensor BF5 (Oji Scientific Instruments)
Flow rate: 1.0 mL/min
Injection volume: 5 μL
Thermostatic bath temperature: 37° C.
Measurement time: 90 seconds
Concentration determination method: Hydrogen peroxide generated by enzymatic degradation of glucose and ethanol was detected, and calculated based on the peak height of a standard solution.
The concentration of residual glucose in the culture broth was shown in FIG. 6 (a) and the concentration of ethanol, a metabolite of glucose, was shown in FIG. 6 (b). Comparing the normal strain and the flocculation strain, the time-course changes in each of the glucose concentration and the ethanol concentration during the cultivation were generally similar.
Comparing the normal strain and the flocculation strain, the time-course changes in each of the OD660 value, the glucose concentration and the ethanol concentration during the cultivation were generally similar. From this, it can be assumed that the glucose metabolism and the cell growth proceeded at approximately the same rate in the normal strain and the flocculation strain by cultivating them under identical conditions. It seems that the cell-growth properties were less affected by the genetic engineering operation for imparting a flocculation property.
[Test Example 9] Confirmation of AaBGL1 Expression by Activity Measurement
By using the above-obtained culture supernatant of the AaBGL1 expression strain, a diluted enzyme sample was prepared, and then the activity was measured in accordance with the following method.
(Activity Measurement Method)
To 10 μl of 20 mM p-nitrophenyl-P-D-glucoside (hereinafter abbreviated as pNPG), 10 μl of 1M sodium acetate buffer solution (pH 4.5) and 130 μl of water were added, and then 50 μl of the diluted enzyme sample was introduced for reacting them at 37° C. for 10 minutes. 100 μl of the reaction mixture was mixed with 100 μl of 2% sodium carbonate solution to terminate reaction, and then the amount of free p-nitrophenol was colorimetrically measured at a wavelength of 450 nm.
The amount of enzyme that produces 1 μmol of p-nitrophenol per minute was defined as 1 U. The pNPG degradation activity (hereinafter also referred to as pNPG activity) per 1 ml of the normal strain or flocculation strain culture supernatant was shown in FIG. 7.
As shown in FIG. 7, the flocculation strain showed an activity value 1.38 times higher than that of the normal strain after cultivating for 15.5 hours and immediately after the depletion of glucose, and showed an activity value 1.27 times higher than that of the normal strain after cultivating for 48 hours.
[Test Example 10] Fed-Batch Culture of a Transformant
The normal strain or the flocculation strain was inoculated in 5 ml of YES medium, and then subjected to pre-culture 1 in a test tube for 24 hours at 32° C. Further, to 200 ml of YES medium, 4 ml of the culture broth obtained by pre-culture 1 was added, and then pre-culture 2 was carried out in a 1 L Sakaguchi flask for 24 hours at 30° C.
Thereafter, by using a 5 L jar fermenter, the culture broth obtained by pre-culture 2 was added into 1,800 ml of an initial culture medium having the composition shown in Table 1, followed by cultivation at 30° C. Here, the concentration of each component in Table 1 indicates the concentration after the inoculation of pre-culture 2. 14.0 hours after starting the cultivation, upon ascertaining that the concentration of residual glucose in the culture broth was lower than 1.0 g/L, feeding was started. The feeding was continued for 81 hours, and 1,450 ml of a feed medium having the composition of Table 2 was added into the jar fermenter, thereby to cultivate for 95 hours (a cultivation time after starting the cultivation) at 30° C. The pH was maintained at 4.5 by controlling the addition of 12.5% ammonia water.










TABLE 1







Component
Concentration




















Yeast extract
10
g/L



Glucose (hydrous)
33
g/L



(NH4)2SO4
15
g/L



KH2PO4
8
g/L



MgSO4•7H2O
5.34
g/L



Na2HPO4
0.04
g/L



CaCl2•2H2O
0.2
g/L



Choline chloride
15.00
mg/L



Folic acid
0.08
mg/L



Pyridoxine
2.16
mg/L



Thiamine
26.49
mg/L



Thymidine
8.75
mg/L



Riboflavin sodium phosphate
7.97
mg/L



p-aminobenzoic acid
38.16
mg/L



Pantothenic acid Ca
15.70
mg/L



Nicotinic acid
49.00
mg/L



Inositol
27.00
mg/L



Biotin
0.08
mg/L



FeC6H5O7•nH2O
28.40
mg/L



ZnSO4•7H2O
25.40
mg/L



MnCl2•4H2O
4.10
mg/L



CuSO4•5H2O
3.80
mg/L



H3BO3
2.90
mg/L



Na2MoO4•2H2O
0.68
mg/L



KI
0.20
mg/L



NiSO4•6H2O
0.44
mg/L



CoCl2•6H2O
0.40
mg/L







(moisture content of glucose: 8.8%)
















TABLE 2







Component
Concentration




















Yeast extract
10
g/L



Glucose (hydrous)
550
g/L



CaCl2•2H2O
0.20
g/L



KH2PO4
9.00
g/L



MgSO4 7H2O
4.45
g/L



K2SO4
3.50
g/L



Na2SO4
0.14
g/L



Na2HPO4
0.04
g/L



Choline chloride
15.00
mg/L



Folic acid
0.08
mg/L



Pyridoxine
2.16
mg/L



Thiamine
26.49
mg/L



Thymidine
8.75
mg/L



Riboflavin sodium phosphate
7.97
mg/L



p-aminobenzoic acid
38.16
mg/L



Pantothenic acid Ca
82.37
mg/L



Nicotinic acid
715.70
mg/L



Inositol
693.70
mg/L



Biotin
0.74
mg/L



FeC6H5O7•nH2O
473.4
mg/L



ZnSO4•7H2O
423.4
mg/L



MnCl2•4H2O
68.4
mg/L



CuSO4•5H2O
63.4
mg/L



H3BO3
48.4
mg/L



Na2MoO4•2H2O
11.4
mg/L



KI
3.4
mg/L



NiSO4•6H2O
7.4
mg/L



CoCl2•6H2O
6.7
mg/L







(moisture content of glucose: 8.8%)






To evaluate the cell growth of the transformant, the absorbance of a culture broth at OD660 nm was measured by a spectrophotometer (Spectrophotometer U-1500). In a case where the concentration of the culture broth is high, dilution with RO water was carried out for the measurement. In the case of measuring the OD660 nm of the flocculation strain, before the measurement, suspension with 100 mM to 500 mM EDTA was carried out for deflocculation.
The results of the cell growth are shown in FIG. 8. Comparing the normal strain and the flocculation strain, the time-course changes in the OD660 values during the cultivation were generally similar, and the final OD660 values of the normal strain and the flocculation strain were found to be 376 and 395, respectively. By using a semi-synthetic culture broth prepared by adding Yeast Extract derived from natural sources into a synthetic culture broth, the high cell concentrations were achieved.
The concentration of residual glucose or a metabolite thereof, ethanol, in the culture broth was measured in the same manner as in Test Example 8.
The concentration of residual glucose in the culture broth was shown in FIG. 9 (a) and the concentration of ethanol, a metabolite of glucose, was shown in FIG. 9 (b). Comparing the normal strain and the flocculation strain, the time-course changes in each of the glucose concentration and the ethanol concentration during the cultivation were generally similar. The glucose concentration remained at a low value of 1 g/L or lower after starting feeding. The ethanol concentration of the culture broth finally remained at a low value of 1 g/L or lower, since ethanol generated at the batch culture stage decreased gradually after starting feeding. After starting feeding, until the end of the fed-batch culture, a glucose effect which triggers an ethanol production phase was not observed.
Comparing the normal strain and the flocculation strain, the time-course changes in each of the OD660 value, the glucose concentration and the ethanol concentration during the cultivation were generally similar. From this, it can be assumed that the glucose metabolism and the cell growth proceeded at approximately the same rate in the normal strain and the flocculation strain by cultivating them under identical conditions. It seems that the cell-growth properties were less affected by the genetic engineering operation for imparting a flocculation property.
[Test Example 11] Measurement of a Sedimentation Rate
Each 1 L of the samples obtained at the end of fed-batch culture in Test Example 10 was transferred to a 1 L graduated cylinder, thereby to compare the sedimentation rate. The cells in each graduated cylinder were suspended sufficiently, and the graduated cylinder was allowed to stand still to start sedimentation. The distance between the liquid surface and the solid-liquid interface (interface between the sedimented yeast cells and the supernatant) was divided by the time elapsed from the onset of sedimentation, thereby to calculate the sedimentation rate. The results are shown in FIG. 10.
As shown in FIG. 10, the sedimentation of the flocculation strain was found to be high as compared with the normal strain. The sedimentation rate of the flocculation strain was 30 mm/h at the time of 2 hours after starting the sedimentation, and was 6.7 mm/h at the time of additional 10 hours (12 hours after starting the sedimentation). On the other hand, the sedimentation rate of the normal strain was 1.5 mm/h at the time of 12 hours after starting the sedimentation.
Further, the results of a microscopic observation (results of trypan blue staining) of the samples obtained at the end of fed-batch culture are shown in FIG. 11. As shown in FIG. 11, from the microscopic observation results of the sample at the end of fed-batch culture, clustering and aggregation of the cells were confirmed in the flocculation strain, disappearance of a flocculation property after fed-batch culture was not observed. In the normal strain, the cells were not aggregated, and were found to be dispersed.
[Test Example 12] Optimization of a Cultivation Temperature (Fed-Batch Culture)
The normal strain (ASP3660 strain) was inoculated in 5 ml of YES medium, and then subjected to pre-culture 1 in a L-shaped test tube for 24 hours at 30° C. Further, to 120 ml of YES medium, 2.4 ml of the culture broth obtained by pre-culture 1 was added, and then pre-culture 2 was carried out in a 500 mL Sakaguchi flask for 24 hours at 30° C.
Thereafter, by using a 3 L jar fermenter, the culture broth obtained by pre-culture 2 was added into 1,080 ml of an initial culture medium, thereby to start cultivation. Cultivation was carried out by using two culture tanks and applying two different temperature conditions of 30° C. and 34° C. As the initial culture medium, a medium prepared by removing Yeast Extract, choline chloride, folic acid, pyridoxine, thiamine, thymidine, riboflavin sodium phosphate and p-aminobenzoic acid from the composition of Table 1 was used. Here, the concentration of each component in Table 1 indicates the concentration after the inoculation of pre-culture 2. 11.8 hours after starting the cultivation, feeding was started. The feeding was continued for 84.2 hours, and 685 ml of a feed medium was added into the jar fermenter, thereby to cultivate for 96 hours (a cultivation time after starting the cultivation) at 30° C. or at 34° C. As the feed medium, a medium prepared by removing Yeast Extract, choline chloride, folic acid, pyridoxine, thiamine, thymidine, riboflavin sodium phosphate and p-aminobenzoic acid from the composition of Table 2 was used. The pH was maintained at 4.5 by controlling the addition of 12.5% ammonia water.
To evaluate the cell growth of the transformant, the OD660 nm value of the culture broth was measured in the same manner as in Test Example 8. The cell growth results are shown in FIG. 12. Comparing the 30° C. condition and the 34° C. condition, the time-course changes in the OD660 values of culture broths were generally similar. The final OD660 values in the 30° C. condition and the 34° C. condition were respectively found to be 295 and 271, whereby the high cell concentrations were achieved.
The concentration of residual glucose or a metabolite thereof, ethanol, in the culture broth was measured in the same manner as in Test Example 8. The concentration of residual glucose in the culture broth was shown in FIG. 13A and the concentration of ethanol, a metabolite of glucose, was shown in FIG. 13B. Comparing the 30° C. condition and the 34° C. condition, the time-course changes in each of the glucose concentration and the ethanol concentration during the cultivation were generally similar. The glucose concentration remained at a low value of 1 g/L or lower after cultivating 24 hours. The ethanol concentration of the culture broth finally remained at a low value of 2 g/L or lower, since ethanol generated at the batch culture stage decreased gradually after starting feeding.
The pNPG degradation activity of the culture broth was measured in the same manner as in Test Example 9. The pNPG degradation activity per 1 ml of the culture supernatant in the 30° C. condition or the 34° C. condition was shown in FIG. 14. In terms of a pNPG activity value, the 34° C. condition showed an activity value 2.26 times higher than that of the 30° C. condition at the end of cultivation after cultivating for 96 hours.
[Test Example 13] Confirmation of AaBGL1 Expression by SDS-PAGE Analysis
Each 5 μl of the culture supernatant samples obtained at end of fed-batch culture in Test Example 12 was dissolved in a SDS-PAGE sample buffer, and SDS-PAGE was carried out with a 4 to 12% acrylamide gel, followed by staining with Coomassie brilliant blue. The results are shown in FIG. 15. From the results of SDS-PAGE, at end of cultivation after cultivating for 96 hours, in the 34° C. condition, the intensity of an AaBGL1 band at a molecular weight of 120 kDa or larger was more intense than the case of the 30° C. condition, whereby the increase in the secretory expression amount of AaBGL1 was observed. Addition of a saccharide chain to AaBGL1 resulted in the detection of smear bands.
[Test Example 14] Protein Production by Hsp9 Promoter
<Preparation of an Expression Vector>
By a PCR method using In-fusion primers and pEGFP-N1 (manufactured by CLONTECH) containing GFP gene as a template, an ORF fragment of EGFP gene was amplified. On the other hand, pSL14 was subjected to double digestion with restriction enzymes AflII and XbaI. The digested fragment and the ORF fragment of EGFP gene amplified by PCR were circularized by In-fusion method, and then introduced into E. coli DH5α (Takara Bio, Inc.) to obtain a transformant. From the obtained transformant, a vector was prepared to obtain a desire expression vector pSL14-EGFP (FIG. 16). The obtained expression vector was confirmed to be a desired vector by restriction enzyme mapping and partial nucleotide sequencing.
In the same manner, pSL6-EGFP vector (FIG. 17) was prepared by integrating the ORF fragment of EGFP into pSL6, and was used as a control.
<Preparation of a Transformant>
As the host cell, a leucine-auxotrophic strain of Schizosaccharomyces pombe (Genotype: h−, leu1-32, provided from professor Yuichi lino, Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo) (ATCC38399) was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of the above-obtained expression vector pSL14-EGFP digested by restriction enzyme NotI was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium.
The transformant obtained three days after cultivation was named as ASP3395 strain.
In the same manner, pSL14-EGFP was introduced to obtain a transformant SL14E strain, and pSL6-EGFP was introduced to obtain a transformant SL6E strain.
<EGFP Expression>
The obtained ASP3395 strain was inoculated in 5 ml of YES medium contained in a test tube, and cultivated for 70 hours at 32° C. At the end of cultivation, the fluorescence intensity of the culture broth excited at 488 nm was measured.
As controls, SL14E strain and SL6E strain were cultivated under the same condition, and the florescence intensities of the culture broths at the end of cultivation were measured.
The measurement results are shown in FIG. 18. As a result, the fluorescence intensity (relative value) of SL14E strain was found to be only 0.50, when the fluorescence intensity of SL6E strain was taken as 1, but the fluorescence intensity (relative value) of ASP3395 strain was found to be 17.17, and was significantly high.
The fluorescence intensity of a culture broth is an index for the expression amount of fluorescent protein EGFP. Since the expression efficiency of a hsp9 promoter in a yeast of the genus Schizosaccharomyces is significantly high, it is apparent that the transformant obtained by using the expression vector of the present invention can produce a much larger amount of a foreign protein than the case of using other promoters.
[Test Example 15] Protein Production by Ihc1 Promoter
<Preparation of a Multiple Cloning Vector>
Single-locus integration type recombination vector pSL17 was prepared by the following steps. Firstly, the hCMV promoter region of publicly known integration type multiple cloning vector pSL6 for fission yeast (FIG. 19, 5,960 bp, SEQ ID NO: 11) was replaced with the promoter of S. pombe ihc1 gene (ihc1 promoter), thereby to prepare multiple cloning vector pSL12 (FIG. 20, 5,847 bp).
Specifically, at first, a region 1 to 501 bp (SEQ ID NO: 9) upstream from the 5′ end (A of initiation codon ATG) of the ORF of S. pombe ihc1 gene was amplified by using genomic DNA derived from a wild-type strain of S. pombe (ARC032 strain, corresponds to ATCC38366, 972h) as a template, a forward primer (ihc1-promoter-F: Table 3) comprising the restriction enzyme recognition site for BlnI at the 5′ end, and a reverse primer (ihc1-promoter-R: Table 3) comprising the restriction enzyme recognition site for KpnI at the 5′ end, thereby to obtain a fragment (ihc1 promoter fragment) having the restriction enzyme recognition site for BlnI at the 5′ end and the restriction enzyme recognition site for KpnI at the 3′ end.
Into a fragment prepared by double digestion of pSL9 with restriction enzymes BlnI and KpnI, a fragment prepared by double digestion of the ihc1 promoter fragment with restriction enzymes BlnI and Kpn was integrated by ligation, thereby to obtain integration type vector pSL12 for fission yeast (SEQ ID NO:14).
Then, the LPI terminator region of pSL12 was replaced with the terminator of S. pombe ihc1 gene (ihc1 terminator), thereby to prepare multiple cloning vector pSL17 (FIG. 21, 5,831 bp).
Specifically, at first, a region 1 to 200 bp (SEQ ID NO: 15) downstream from the 3′ end (the third letter of stop codon) of the ORF of S. pombe ihc1 gene was amplified by using genomic DNA derived from a wild-type strain of S. pombe (ARC032 strain, corresponds to ATCC38366, 972h−) as a template and In-fusion primers (ihc1-terminator-F and ihc1-terminator-R: Table 3), thereby to obtain a fragment having the ihc terminator region (ihc terminator fragment).
By using pSL12 as a template, PCR amplification was carried out with In-fusion primers (pSL12-F and pSL12-R: Table 3) to obtain a pSL12 full-length fragment lacking the LPI terminator region. Then, into thus obtained fragment, the ihc terminator fragment was integrated by using In-fusion cloning kit (product name: In-Fusion HD Cloning Kit w/Cloning Enhancer, Takara Bio Inc.), thereby to prepare multiple cloning vector pSL17 (SEQ ID NO: 20).









TABLE 3






Sequence
SEQ ID NO.







ihc1-promoter-F
5′-AATTCCTAGGGGCTTCAACTAGCTCGGGAT-3′
12


 


ihc1-promoter-R
5′-AATTGGTACCGACACCTGCTTCACATTGTTT
13



ATAAATAAAATTGGAGTT-3′



 


ihc1-terminator-F
5′-CCTGCAGGCATGCAAGCTTATTGCTGCCCAG
16



TTGATTACCC-3′



 


ihc1-terminator-R
5′-GAAACGCGCGAGGCAGATCATTGTATGCTAT
17



GGGGTGTCGAC-3′



 


pSL12-F
5′-GTCGACACCCCATAGCATACAATGATCTGCC
18



TCGCGCGTTTC-3′



 


pSL12-R
5′-GGGTAATCAACTGGGCAGCAATAAGCTTGCA
19



TGCCTGCAGG-3′









<Preparation of an Expression Vector>

By a PCR method using In-fusion primers and pEGFP-N1 (manufactured by CLONTECH) containing GFP gene as a template, an ORF fragment of EGFP gene was amplified. On the other hand, the above-described pSL12 was subjected to double digestion with restriction enzymes AfIII and XbaI. The digested fragment and the ORF fragment of EGFP gene amplified by PCR were circularized by In-fusion method, and then introduced into E. coli DH5α (Takara Bio, Inc.) to obtain a transformant. From the obtained transformant, a vector was prepared to obtain a desired expression vector pSL12-EGFP (FIG. 22). The obtained expression vector was confirmed to be a desired vector by restriction enzyme mapping and partial nucleotide sequencing.
In the same manner, pSL6-EGFP vector (FIG. 17) was prepared by integrating the ORF fragment of EGFP into pSL6, and was used as a control.
<Preparation of a Transformant>
As the host cell, a leucine-auxotrophic strain of Schizosaccharomyces pombe (Genotype: h−, leu1-32, provided from professor Yuichi lino, Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo) (ATCC38399) was cultivated in YES medium (0.5% of yeast extract, 3% of glucose and 0.1 mg/ml of SP supplements) until 0.6×107 cells/ml. The cells were collected and washed, and then suspended by 0.1M lithium acetate (pH 5.0) to 1.0×108 cells/ml. Thereafter, to 100 μl of the suspension, 1 μg of the above-obtained expression vector pSL12-EGFP digested by restriction enzyme NotI was added, and then 290 μl of a 50% (w/v) polyethylene glycol (PEG4000) aqueous solution was added thereto, followed by stirring to incubate them for 60 minutes at 30° C., 5 minutes at 42° C., and 10 minutes at room temperature, in this order. PEG4000 was removed by centrifugation and then the cells were washed to suspend them in 150 μl of sterile water. The suspension was applied on a minimal-agarose medium.
The transformant obtained three days after cultivation was named as 277G strain.
In the same manner, pSL6-EGFP was introduced to obtain a transformant of SL6E strain.
<EGFP Expression>
The obtained 277G strain was inoculated in 5 ml of YES medium contained in a test tube, and cultivated for 72 hours at 32° C. From the start to the end of cultivation, the fluorescence intensity excited at 488 nm and the absorbance at 660 nm of the culture broth were measured over time.
As a control, SL6E strain was cultivated under the same condition, and the florescence intensity and the absorbance at 660 nm of the culture broth were measured over time.
The time-course changes in [fluorescence-intensity/OD600] (a value obtained after dividing the fluorescence intensity by the observance at 600 nm) of the culture broth of each strain are shown in FIG. 23. As a result, the [fluorescence-intensity/ODD600] of 277G strain (“ihc1p” in Figure) was found to be lower than that of SL6E strain using hCMV promoter (“hCMVp” in Figure) at the time of 24 hours after cultivation, but was found to be higher than that of SL6E strain after cultivating 48 hours, whereby the EGFP expression amount per single cell was found to be high.
This application is a continuation of PCT Application No. PCT/JP2013/072195, filed on Aug. 20, 2013, which is based upon and claims the benefit of priority from Japanese Patent Application No. 2012-181865 filed on Aug. 20, 2012. The contents of those applications are incorporated herein by reference in its entirety.

## Claims

1. A cloning vector comprising the nucleotide sequence of SEQ ID NO: 9 comprising a SPAC22G7.11c gene promoter and a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence comprises a cloning site located downstream from the promoter and a terminator capable of functioning in a yeast of the genus Schizosaccharomyces, wherein the promoter is capable of regulating expression of a heterologous gene introduced into the cloning site.

2. A method for producing an expression vector, comprising introducing a heterologous gene into the cloning site of the cloning vector of claim 1.

3. An expression vector comprising the cloning vector of claim 1 further comprising a heterologous gene which is introduced into the cloning site.

4. A method comprising introducing the expression vector of claim 3 into a yeast of the genus Schizosaccharomyces.

5. A transformant of a yeast of the genus Schizosaccharomyces, wherein the transformant comprises the expression vector of claim 3.

6. A method comprising cultivating the transformant of claim 5, expressing a protein encoded by the heterologous gene, and recovering the protein.